(4) Dac(i)=p/(p+q) Dma+b (fa(i)/(1-fa(i)))1/ma+b # (5) Dbc(i)=q/(p+q) Dma+b (fa(i)/(1-fa(i)))1/ma+b # (6) Molar ratio p/q = Dm(a) /Dm(b) = 10/30 = 1/3

Obtained from linear regression of experimental data

computed using the formulas for standard error (SE, eq. 13 and eq. 14).

CI(i) = Dac(i) /Das(i) +Dbc(i) /Dbs(i) + α Dac(i) . Dbc(i) / Das(i) . Dbs(i)

Table 5. CI calculation between two drugs a and b at the 50% effect level under three alternative conditions: additive, synergic, antagonistic. To obtain CI as a function of the effect level i, the calculation has to be repeated for each arbitrary level i between 0 and 1. A 95% confidence interval around D in general and around Dm in particular, can be computed using the formulas for standard error (SE, eq. 13 and eq. 14). A 95% confidence interval around CI at any effect level i can be computed from the standard error formulas presented

Single drug b

**Additive Synergic Antagonistic**

Combined drugs a+b

+{ Dbc(i)/ Dbs(i) .

+[SE(m)/m] 2+2[-(logD)1/2 . SE(m)/SE/(b)] .

Combined drugs a+b

**Three alternative results of the experimental assay with combination** *a* **+***b (* **considering α=1, mutually non-exclusive condition)**

Table 5 summarizes a manual calculation of CI of two drugs a and b using these formulas for the 50% effect level under three hypothetical results: additive, synergic or antagonistic effect.

The same calculation can be applied to any effect level to plot CI as a function of effect level. When the interaction is additive CI =1. In this case it can be interpreted that one of the drugs (the less potent one, i.e. drug b in the example) is acting as though it is merely a diluted form of the other (drug a in the example). When CI<1 the combination of a+b is synergic while CI>1 indicates antagonism. Synergy, implies that the combination of the two drugs achieves a cytotoxic effect greater than that expected by the simple addition of the effects of the drugs a and b, while antagonism achieves a cytotoxic effect lower than that expected by additive effects of drugs a and b.

Fig. 5. Drug interaction and CI calculation. A. Only rarely the combination index obtained is constant for all effect levels. Here it is shown how different values of the slope m obtained through linear regression in the combination experiment (a+b) would affect the shape of the curve representing the CI as a function of the effect level. Similarly, differences between the slopes obtained for drugs a and b through the single drug experiments will contribute to the uneven shape of the CI function. Note that depending on the effect level the interaction a+b with m=1.8 would be synergism, additive or antagonism at EC25. EC50, and EC75 respectively (arrows). B. Results of CI calculation for the example where a+b results in synergism considering m=4 in the regression of the combined-drug experiment. C. Results of CI calculation for the example where a+b results in antagonism considering m=2.5 in the regression of the combined-drug experiment. D. 95% confidence level intervals around CI, using an algebraic approximation (eq. 12, Table 5) in an example where combination of a+b is synergic

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The horizontal line corresponding to CI(i)=1, where i is any effect level in the interval (0,1), is often call the additive effect line. A combination of drugs a and b may result in CI values above or below the additive line at different effect levels. Thus, the CI as a function of effect level is not constant or linear and it may be decreasing or increasing (Fig 5A). If data from the combination experiment in the example of figure 4A resulted in ma+b=5 or ma+b=1.8, even still with Dma+b =17 the CI line would be inclined downwards or upwards respectively (Fig. 5A). Only at effect levels close to EC50 the result would be strictly additive. An important conclusion is that for some drug combinations, experiments conducted at different single dose-effect levels may yield opposing results. For example if the combination a+b with m=1.8 shown in figure 5A were experimentally evaluated only at effect level 0.25 the single dose analysis would conclude on synergism. However if it were evaluated at effect level 0.75 it would conclude on antagonism (Fig. 5A). This exemplifies why the assessment of combination index over the whole dose range will show all kinds of interactions that may result from combination at different effect levels.

Computing a standard error of CI allows plotting confidence intervals at all effect levels providing a further assurance over the computation. A 95% confidence interval will indicate that if we repeat the experiment 100 times, 95 out 100 times the CI would be within this interval. For example, observing whether or not confidence limits are above or below the additive line will allow concluding with further statistical support on antagonism or synergism respectively. Computation of the standard error of CI and confidence intervals at all levels should be better obtained through specialized software such as Calcusyn or Compusyn (Bijnsdorp et al., 2011; Chou, 2010). It may also require approaches such as Monte Carlo simulation based on the estimated parameter for m and Dm in single and combined equations.

#### **4. Concluding remarks**

A thorough assessment of drug interaction is an essential step in targeted combined therapy. The new targeted agents are seldom useful as single agents but may be effective when used in specific combinations. The median effect and combination index calculation are well founded methods traditionally used in pharmacological and toxicological studies. Since new cytotoxic drugs target mechanisms eliciting cell death, biomarkers related to viability assessment are preferred to biomarkers of cell proliferation. Flow cytometry is an ideal technology to provide massive data from cell death biomarkers to build dose response curves of cytotoxic effect. When these data is further used to determine the combination index a full characterization of drug interaction over the cytotoxic effect is obtained at all effect levels. This approach can be applied to tumor cell lines in preclinical studies and also in patient-derived tumor cells, thus providing useful information as prospective indicators of the potential therapeutic response to combined-drug antitumor treatment.

#### **5. References**

Armand, J. P., Burnett, A. K., Drach, J., Harousseau, J. L., Lowenberg, B. & San Miguel, J. (2007). The emerging role of targeted therapy for hematologic malignancies: update on bortezomib and tipifarnib. Oncologist Vol. 12, No. 3, (Mar, 2007), pp. 281-290

The horizontal line corresponding to CI(i)=1, where i is any effect level in the interval (0,1), is often call the additive effect line. A combination of drugs a and b may result in CI values above or below the additive line at different effect levels. Thus, the CI as a function of effect level is not constant or linear and it may be decreasing or increasing (Fig 5A). If data from the combination experiment in the example of figure 4A resulted in ma+b=5 or ma+b=1.8, even still with Dma+b =17 the CI line would be inclined downwards or upwards respectively (Fig. 5A). Only at effect levels close to EC50 the result would be strictly additive. An important conclusion is that for some drug combinations, experiments conducted at different single dose-effect levels may yield opposing results. For example if the combination a+b with m=1.8 shown in figure 5A were experimentally evaluated only at effect level 0.25 the single dose analysis would conclude on synergism. However if it were evaluated at effect level 0.75 it would conclude on antagonism (Fig. 5A). This exemplifies why the assessment of combination index over the whole dose range will show all kinds of interactions that may

Computing a standard error of CI allows plotting confidence intervals at all effect levels providing a further assurance over the computation. A 95% confidence interval will indicate that if we repeat the experiment 100 times, 95 out 100 times the CI would be within this interval. For example, observing whether or not confidence limits are above or below the additive line will allow concluding with further statistical support on antagonism or synergism respectively. Computation of the standard error of CI and confidence intervals at all levels should be better obtained through specialized software such as Calcusyn or Compusyn (Bijnsdorp et al., 2011; Chou, 2010). It may also require approaches such as Monte Carlo simulation based on the estimated parameter for m and Dm in single and

A thorough assessment of drug interaction is an essential step in targeted combined therapy. The new targeted agents are seldom useful as single agents but may be effective when used in specific combinations. The median effect and combination index calculation are well founded methods traditionally used in pharmacological and toxicological studies. Since new cytotoxic drugs target mechanisms eliciting cell death, biomarkers related to viability assessment are preferred to biomarkers of cell proliferation. Flow cytometry is an ideal technology to provide massive data from cell death biomarkers to build dose response curves of cytotoxic effect. When these data is further used to determine the combination index a full characterization of drug interaction over the cytotoxic effect is obtained at all effect levels. This approach can be applied to tumor cell lines in preclinical studies and also in patient-derived tumor cells, thus providing useful information as prospective indicators

Armand, J. P., Burnett, A. K., Drach, J., Harousseau, J. L., Lowenberg, B. & San Miguel, J.

(2007). The emerging role of targeted therapy for hematologic malignancies: update on bortezomib and tipifarnib. Oncologist Vol. 12, No. 3, (Mar, 2007), pp. 281-290

of the potential therapeutic response to combined-drug antitumor treatment.

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combined equations.

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Wlodkowic, D. & Skommer, J. (2007). SYTO probes: markers of apoptotic cell demise. Curr

Woodcock, J., Griffin, J. P. & Behrman, R. E. (2011). Development of novel combination therapies. N Engl J Med Vol. 364, No. 11, (Mar 17, 2011), pp. 985-987 Workman, P., Burrows, F., Neckers, L. & Rosen, N. (2007). Drugging the cancer chaperone

Wright, J. J. (2010). Combination therapy of bortezomib with novel targeted agents: an

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**21** 

*Czech Republic* 

**Flow Cytometry Analysis of Intracellular Protein** 

The technique of measuring intracellular protein levels using flow cytometry is a very rapid

The demand for determining protein levels in a continuously decreasing amount of input cells is currently being developed by scientists and medical doctors. Another demanding area is the splitting of active and inactive (phosphorylated and unphosphorylated, respectively) forms of the protein being monitored, e.g., during different phases of the cell cycle, differentiation or carcinogenesis. The Western blot technique has been typically used for this purpose. The utilization of multi color flow cytometry allows for measurements of multiple proteins in parallel, regardless of the protein length. As the technique evolves, it is possible to obtain information on over 13 parameters per cell

Measuring the phosphorylation status of specific proteins using this technique is very efficient. It eliminates a number of problems related either to Western blotting or ELISA.

Compared to the Western blot, this progressive method has much lower requirements on the number of input cells (only 1 x 104 cells are needed for determining the concentration of a given protein), it requires less time for processing and, last but not least, is also much more cost effective. Furthermore, it allows researchers to distinguish the various cell populations in the sample without complicated cell separation, if the appropriate antibodies are used. This is beneficial for searching for cell populations and their reactions on external stimuli, such as changed cultivation conditions during in vitro experiments or influencing the

In this chapter, the application and pitfalls of flow cytometric intracellular protein measurements are illustrated by the data of our original research of porcine monocytes and CD34+ hematopoietic stem/progenitor cells. In short, a successful utilization of this

1. Precise experimental planning to obtain a defined cell population using this technique.

progressive technique depends on correctly performing the 4 following steps:

2. Effective fixation and permeabilization of the cell population.

4. Selecting the optimal system of primary and secondary antibodies.

**1. Introduction** 

(Krutzik et al., 2004).

organism during in vivo experiments.

3. Choosing the optimal isotype control.

method to detect protein on a single-cell level.

Irena Koutná1, Pavel Šimara1, Petra Ondráčková2 and Lenka Tesařová1

*Masaryk University/ Centre for Biomedical Image Analysis, FI Veterinary Reasearch Institute/ Department of Immunology* 


### **Flow Cytometry Analysis of Intracellular Protein**

Irena Koutná1, Pavel Šimara1, Petra Ondráčková2 and Lenka Tesařová1 *Masaryk University/ Centre for Biomedical Image Analysis, FI Veterinary Reasearch Institute/ Department of Immunology Czech Republic* 

#### **1. Introduction**

420 Flow Cytometry – Recent Perspectives

Zanetti, M., d'Uscio, L. V., Peterson, T. E., Katusic, Z. S. & O'Brien, T. (2005). Analysis of

Zhang, N., Wu, Z. M., McGowan, E., Shi, J., Hong, Z. B., Ding, C. W., Xia, P. & Di, W. (2009).

65-72

(Dec, 2009), pp. 2459-2464

superoxide anion production in tissue. Methods Mol Med Vol. 108, No., 2005), pp.

Arsenic trioxide and cisplatin synergism increase cytotoxicity in human ovarian cancer cells: therapeutic potential for ovarian cancer. Cancer Sci Vol. 100, No. 12,

> The technique of measuring intracellular protein levels using flow cytometry is a very rapid method to detect protein on a single-cell level.

> The demand for determining protein levels in a continuously decreasing amount of input cells is currently being developed by scientists and medical doctors. Another demanding area is the splitting of active and inactive (phosphorylated and unphosphorylated, respectively) forms of the protein being monitored, e.g., during different phases of the cell cycle, differentiation or carcinogenesis. The Western blot technique has been typically used for this purpose. The utilization of multi color flow cytometry allows for measurements of multiple proteins in parallel, regardless of the protein length. As the technique evolves, it is possible to obtain information on over 13 parameters per cell (Krutzik et al., 2004).

> Measuring the phosphorylation status of specific proteins using this technique is very efficient. It eliminates a number of problems related either to Western blotting or ELISA.

> Compared to the Western blot, this progressive method has much lower requirements on the number of input cells (only 1 x 104 cells are needed for determining the concentration of a given protein), it requires less time for processing and, last but not least, is also much more cost effective. Furthermore, it allows researchers to distinguish the various cell populations in the sample without complicated cell separation, if the appropriate antibodies are used. This is beneficial for searching for cell populations and their reactions on external stimuli, such as changed cultivation conditions during in vitro experiments or influencing the organism during in vivo experiments.

> In this chapter, the application and pitfalls of flow cytometric intracellular protein measurements are illustrated by the data of our original research of porcine monocytes and CD34+ hematopoietic stem/progenitor cells. In short, a successful utilization of this progressive technique depends on correctly performing the 4 following steps:


Flow Cytometry Analysis of Intracellular Protein 423

The fixation and permeabilization process could damage the light scatter properties of the cells. The light scatter characteristics of all tested cells were comparable after any type of fixation and permeabilization. The light scatter characteristics were always changed in comparison with fresh preparations. However, well distinguishable and bounded subpopulations of mononuclear cells and neutrophils were obtained by adjusting the

Some protocols use labeling of intracellular molecules in the whole blood instead of isolated blood leukocytes. In our laboratory, the lysis of red blood cells (RBC) is always performed before starting the staining procedure. However, in order to make the processing of large amounts of samples as quick as possible, the hemolysis is commonly performed in the 96 well plate in which the cultivation was previously performed. The volume of the hemolytic reagent is therefore relatively small and the hemolysis is not complete. Therefore, it is advantageous if the fixation and permeabilization procedure leads to the lysis of these contaminating erythrocytes. It was found that only the IntraStain kit and paraformaldehyde/saponin fixation and permeabilization completely lysed RBC in the samples. The other two kits did not induce a lysis of porcine RBC (although complete lysis of human RBC was obtained by these kits in preliminary experiments, which is in

The problem can occur when the lysis of RBC is performed before fixation and permeabilization. The lysis of RBC induced by ammonium chloride solution (the lysing reagent commonly used in our laboratory to lyse porcine RBC) before fixation and permeabilization with commercial kits strongly changed the light scatter characteristics of white blood cells (WBC). This was apparent, especially in neutrophils, which completely fused with the lymphocyte population. Moreover, the population of lymphocytes was much more dispersed as shown by the side scatter measurements. If the lysis of RBC was performed before their fixation and permeabilization with the IntraStain kit, and the cells were washed twice in CWS solution immediately after lysis, the scatters were not altered. If these washing steps were tested with the other two kits, the light scatters were changed. Thus, the lysis of RBC cannot be achieved with the Fix&Perm or Leukoperm kits without

Finally, the effect of different fixation/permeabilization reagents on IFN-γ and TNF-α staining was tested. Generally, we can say that commercial kits mostly gave better results compared with paraformaldehyde/saponin. IntraStain and Leukoperm gave better results than Fix&Perm in some cases. If the use of a combination of paraformaldehyde/saponin is considered, 0.05% saponin should be avoided, especially in combination with 2% paraformaldehyde. Saponin in a concentration of 0.1% in combination with 4% paraformaldehyde slightly increased the percentages of positive cells in comparison with

Consistent with the above mentioned parameters, the IntraStain kit gave the best results compared to the other two tested kits, as well as when compared to paraformaldehyde/saponin. Therefore, this kit was chosen for fixation and permeabilization

Since a wide range of different fixation and permeabilization reagents that were not tested in our study are currently available, the above mentioned parameters can serve as a particular

2% paraformaldehyde. However, these differences were nonsignificant.

settings in the side and forward scatters.

accordance with the manufacturer's instructions).

alteration of light scatter characteristics of WBC.

of porcine leukocytes for experimentation.

### **2. Fixation and permeabilization**

Intracellular flow cytometry, in comparison with conventional cell surface labeling methods, requires fixation and permeabilization of the cells before staining of intracellular antigens (Robinson et al., 1993). Moreover, a variety of commercial kits for fixation and permeabilization are available.

The first step in the population analysis is high quality fixation. A crosslinking reagent (typically formaldehyde) could be used for this purpose. For every given cell population, it is necessary to test the adequate concentration of the solution. The most common formaldehyde concentration is about 4%. Other fixation limiting factors are time and temperature. The incubation time, according to the cell population type, varies between 8 and 15 minutes. The incubation temperature is 37 °C. A variety of commercial kits for fixation and permeabilization are available.

The following step is for permeabilization with detergents (Triton X 100 or saponin) or alcohol (ethanol or methanol). Although the protocols that have been used to stain phosphoepitopes for flow cytometry differ from one to another, they rely on two primary permeabilization reagents – saponin or methanol.

#### **Saponin permeabilization**

Saponin is a mixture of terpenoid molecules and glycosides that permeabilize cells by interacting with cholesterol present in the cell membrane (Melan et al. 1999). This creates pores in the plasma membrane that are large enough for entry of fluorophore-conjugated antibodies. Because the intracellular proteins can leak out of saponin-treated cells, they must be first exposed to a crosslinking reagent, such as formaldehyde, to cross-link proteins and nucleic acids into a cohesive unit within the cell. Saponin has become the detergent of choice for cytokine staining, and several groups have utilized it for permeabilization in phosphoepitope staining protocols (Pala et al., 2000). It is typically used at concentrations ranging from 0.1% to 0.5%, similar to cytokine-staining procedures.

Three commercially available kits (Leukoperm, Serotec, UK; Fix & Perm, An Der Grub, Austria; IntraStain, DAKO Cytomation, Denmark) along with combinations of 2 or 4% paraformaldehyde with 0.1 or 0.05% saponin were tested for fixation and permeabilization of isolated pig's peripheral blood mononuclear cells or whole blood leukocytes (Zelnickova et al., 2007).

The fixation and permeabilization process could lead to non-specific binding of primary or secondary antibodies. In comparison to all three tested commercial kits, a combination of paraformaldehyde and saponin caused an increase in non-specific binding of antibodies. The intensity of fluorescence of the negative peak of paraformaldehyde/saponin fixed cells was evidently higher in comparison with the negative peak of cells fixed with the use of commercial kits.

The fixation and permeabilization process could lead to elevation of autofluorescence of cells. The autofluorescence of cells was at the lowest level in all tested kits. In contrast, the combination of paraformaldehyde and saponin in all concentrations caused an increase of autofluorescence. The autofluorescence in samples fixed with 4% paraformaldehyde was higher than with 2% paraformaldehyde.

Intracellular flow cytometry, in comparison with conventional cell surface labeling methods, requires fixation and permeabilization of the cells before staining of intracellular antigens (Robinson et al., 1993). Moreover, a variety of commercial kits for fixation and

The first step in the population analysis is high quality fixation. A crosslinking reagent (typically formaldehyde) could be used for this purpose. For every given cell population, it is necessary to test the adequate concentration of the solution. The most common formaldehyde concentration is about 4%. Other fixation limiting factors are time and temperature. The incubation time, according to the cell population type, varies between 8 and 15 minutes. The incubation temperature is 37 °C. A variety of commercial kits for

The following step is for permeabilization with detergents (Triton X 100 or saponin) or alcohol (ethanol or methanol). Although the protocols that have been used to stain phosphoepitopes for flow cytometry differ from one to another, they rely on two primary

Saponin is a mixture of terpenoid molecules and glycosides that permeabilize cells by interacting with cholesterol present in the cell membrane (Melan et al. 1999). This creates pores in the plasma membrane that are large enough for entry of fluorophore-conjugated antibodies. Because the intracellular proteins can leak out of saponin-treated cells, they must be first exposed to a crosslinking reagent, such as formaldehyde, to cross-link proteins and nucleic acids into a cohesive unit within the cell. Saponin has become the detergent of choice for cytokine staining, and several groups have utilized it for permeabilization in phosphoepitope staining protocols (Pala et al., 2000). It is typically used at concentrations ranging

Three commercially available kits (Leukoperm, Serotec, UK; Fix & Perm, An Der Grub, Austria; IntraStain, DAKO Cytomation, Denmark) along with combinations of 2 or 4% paraformaldehyde with 0.1 or 0.05% saponin were tested for fixation and permeabilization of isolated pig's peripheral blood mononuclear cells or whole blood leukocytes (Zelnickova

The fixation and permeabilization process could lead to non-specific binding of primary or secondary antibodies. In comparison to all three tested commercial kits, a combination of paraformaldehyde and saponin caused an increase in non-specific binding of antibodies. The intensity of fluorescence of the negative peak of paraformaldehyde/saponin fixed cells was evidently higher in comparison with the negative peak of cells fixed with the use of

The fixation and permeabilization process could lead to elevation of autofluorescence of cells. The autofluorescence of cells was at the lowest level in all tested kits. In contrast, the combination of paraformaldehyde and saponin in all concentrations caused an increase of autofluorescence. The autofluorescence in samples fixed with 4% paraformaldehyde was

**2. Fixation and permeabilization** 

fixation and permeabilization are available.

permeabilization reagents – saponin or methanol.

from 0.1% to 0.5%, similar to cytokine-staining procedures.

permeabilization are available.

**Saponin permeabilization** 

et al., 2007).

commercial kits.

higher than with 2% paraformaldehyde.

The fixation and permeabilization process could damage the light scatter properties of the cells. The light scatter characteristics of all tested cells were comparable after any type of fixation and permeabilization. The light scatter characteristics were always changed in comparison with fresh preparations. However, well distinguishable and bounded subpopulations of mononuclear cells and neutrophils were obtained by adjusting the settings in the side and forward scatters.

Some protocols use labeling of intracellular molecules in the whole blood instead of isolated blood leukocytes. In our laboratory, the lysis of red blood cells (RBC) is always performed before starting the staining procedure. However, in order to make the processing of large amounts of samples as quick as possible, the hemolysis is commonly performed in the 96 well plate in which the cultivation was previously performed. The volume of the hemolytic reagent is therefore relatively small and the hemolysis is not complete. Therefore, it is advantageous if the fixation and permeabilization procedure leads to the lysis of these contaminating erythrocytes. It was found that only the IntraStain kit and paraformaldehyde/saponin fixation and permeabilization completely lysed RBC in the samples. The other two kits did not induce a lysis of porcine RBC (although complete lysis of human RBC was obtained by these kits in preliminary experiments, which is in accordance with the manufacturer's instructions).

The problem can occur when the lysis of RBC is performed before fixation and permeabilization. The lysis of RBC induced by ammonium chloride solution (the lysing reagent commonly used in our laboratory to lyse porcine RBC) before fixation and permeabilization with commercial kits strongly changed the light scatter characteristics of white blood cells (WBC). This was apparent, especially in neutrophils, which completely fused with the lymphocyte population. Moreover, the population of lymphocytes was much more dispersed as shown by the side scatter measurements. If the lysis of RBC was performed before their fixation and permeabilization with the IntraStain kit, and the cells were washed twice in CWS solution immediately after lysis, the scatters were not altered. If these washing steps were tested with the other two kits, the light scatters were changed. Thus, the lysis of RBC cannot be achieved with the Fix&Perm or Leukoperm kits without alteration of light scatter characteristics of WBC.

Finally, the effect of different fixation/permeabilization reagents on IFN-γ and TNF-α staining was tested. Generally, we can say that commercial kits mostly gave better results compared with paraformaldehyde/saponin. IntraStain and Leukoperm gave better results than Fix&Perm in some cases. If the use of a combination of paraformaldehyde/saponin is considered, 0.05% saponin should be avoided, especially in combination with 2% paraformaldehyde. Saponin in a concentration of 0.1% in combination with 4% paraformaldehyde slightly increased the percentages of positive cells in comparison with 2% paraformaldehyde. However, these differences were nonsignificant.

Consistent with the above mentioned parameters, the IntraStain kit gave the best results compared to the other two tested kits, as well as when compared to paraformaldehyde/saponin. Therefore, this kit was chosen for fixation and permeabilization of porcine leukocytes for experimentation.

Since a wide range of different fixation and permeabilization reagents that were not tested in our study are currently available, the above mentioned parameters can serve as a particular

Flow Cytometry Analysis of Intracellular Protein 425

looked up in a company product list according to the desired isotype (IgG1, IgG2a, IgM, etc.), reactivity (mouse, human, rat, etc.), and conjugate (FITC, PE, APC, etc.). In addition, recommended isotype controls can often be found on the Data Sheets for primary

Fig. 1. The difference between permeabilization protocols using (A) methanol and (B) the BD Fix&Perm kit. Permebilization with methanol is more sensitive and allows researchers to distinguish between individual MFI peaks. Fix&Perm kit is less time consuming with

relatively low sensitivity

protocol of intracellular cytokine detection and also as a suggestion for optimization of the fixation, permeabilization and cell surface labeling procedures for any laboratory.

#### **2.1 Methanol permeabilization**

Alcohol permeabilization has typically been used for the analysis of DNA by flow cytometry (Ormerod et al., 2002), but can be successfully applied to phospho-epitope staining as well (Krutzik et al., 2003). It is thought that alcohols fix and permeabilize cells by dehydrating them and solubilizing molecules out of the plasma membrane. Proteins may be made more accessible to antibodies during the process and cells are permeabilized to a greater extent than with saponin, allowing efficient access to the nuclear antigens.

Another option is to use commercially available kits. Currently, there are a large number of them available on the market. A fact that needs to be taken into account when using the commercial kits is that even if the kit works well, the method cannot be excessively modified. This could be reflected in the scale of results.

By using one of the best available kits for permeabilization, BD Fix&Perm, with methanol permeabilization during "The decrease in p-CrkL levels upon imatinib treatment" experiment, we came to the following conclusion:

The method (permeabilization using BD Fix&Perm) is less sensitive. It does not recognize the difference between 0 µM and 5 µM imatinib (IM). The speed (2 hours) is an advantage and there is a smaller amount of necessary input material (cells) as well. Although the peaks are sharp, it seems to be more difficult for the antigens to get into the cell, because of less aggressive permeabilization. The methanol permeabilization is much more sensitive. It is able to recognize differences between 0 µM and 5 µM IM. It is more time consuming (5 hours) and requires a higher amount of input material (cells). The significant advantage is the customizability of the method according to user needs (Figure 1).

#### **3. Isotype control**

The selection of the appropriate isotype control is an important element in flow cytometry experiments. Isotype controls are antibodies of the same isotype as the target primary antibody. They are of unknown specificity or are raised against antigens known to be absent in target cells. Isotype controls are used to estimate non-specific staining of primary antibodies. Several factors can contribute to the levels of this "background" staining, including Fc receptor binding, non-specific protein interactions, and cell autofluorescence. These factors may vary depending on the target cell type and the isotype of the primary antibody. Therefore, isotype controls need to be properly chosen. Isotype control antibodies ideally match the primary antibody's host species, isotype, and conjugation format. For example, if the primary antibody is an APC-conjugated mouse IgG2a, then it will be necessary to choose an APC-conjugated mouse IgG2a isotype control. Thus, isotype control is supposed to have all the non-specific characteristics of the target primary antibody and it is able to accurately determine the level of specific staining. Various monoclonal antibody idiotypes are used in flow cytometry applications: most frequently, IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA; less frequently IgD, IgE, IgG2c, Ig kappa, and Ig lambda are applied. Designing the experiment, this isotype and origin species of the primary antibody must be known to find a suitable isotype control. The appropriate isotype control is subsequently

protocol of intracellular cytokine detection and also as a suggestion for optimization of the

Alcohol permeabilization has typically been used for the analysis of DNA by flow cytometry (Ormerod et al., 2002), but can be successfully applied to phospho-epitope staining as well (Krutzik et al., 2003). It is thought that alcohols fix and permeabilize cells by dehydrating them and solubilizing molecules out of the plasma membrane. Proteins may be made more accessible to antibodies during the process and cells are permeabilized to a greater extent

Another option is to use commercially available kits. Currently, there are a large number of them available on the market. A fact that needs to be taken into account when using the commercial kits is that even if the kit works well, the method cannot be excessively

By using one of the best available kits for permeabilization, BD Fix&Perm, with methanol permeabilization during "The decrease in p-CrkL levels upon imatinib treatment"

The method (permeabilization using BD Fix&Perm) is less sensitive. It does not recognize the difference between 0 µM and 5 µM imatinib (IM). The speed (2 hours) is an advantage and there is a smaller amount of necessary input material (cells) as well. Although the peaks are sharp, it seems to be more difficult for the antigens to get into the cell, because of less aggressive permeabilization. The methanol permeabilization is much more sensitive. It is able to recognize differences between 0 µM and 5 µM IM. It is more time consuming (5 hours) and requires a higher amount of input material (cells). The significant advantage is

The selection of the appropriate isotype control is an important element in flow cytometry experiments. Isotype controls are antibodies of the same isotype as the target primary antibody. They are of unknown specificity or are raised against antigens known to be absent in target cells. Isotype controls are used to estimate non-specific staining of primary antibodies. Several factors can contribute to the levels of this "background" staining, including Fc receptor binding, non-specific protein interactions, and cell autofluorescence. These factors may vary depending on the target cell type and the isotype of the primary antibody. Therefore, isotype controls need to be properly chosen. Isotype control antibodies ideally match the primary antibody's host species, isotype, and conjugation format. For example, if the primary antibody is an APC-conjugated mouse IgG2a, then it will be necessary to choose an APC-conjugated mouse IgG2a isotype control. Thus, isotype control is supposed to have all the non-specific characteristics of the target primary antibody and it is able to accurately determine the level of specific staining. Various monoclonal antibody idiotypes are used in flow cytometry applications: most frequently, IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA; less frequently IgD, IgE, IgG2c, Ig kappa, and Ig lambda are applied. Designing the experiment, this isotype and origin species of the primary antibody must be known to find a suitable isotype control. The appropriate isotype control is subsequently

fixation, permeabilization and cell surface labeling procedures for any laboratory.

than with saponin, allowing efficient access to the nuclear antigens.

the customizability of the method according to user needs (Figure 1).

modified. This could be reflected in the scale of results.

experiment, we came to the following conclusion:

**2.1 Methanol permeabilization** 

**3. Isotype control** 

looked up in a company product list according to the desired isotype (IgG1, IgG2a, IgM, etc.), reactivity (mouse, human, rat, etc.), and conjugate (FITC, PE, APC, etc.). In addition, recommended isotype controls can often be found on the Data Sheets for primary

Fig. 1. The difference between permeabilization protocols using (A) methanol and (B) the BD Fix&Perm kit. Permebilization with methanol is more sensitive and allows researchers to distinguish between individual MFI peaks. Fix&Perm kit is less time consuming with relatively low sensitivity

Flow Cytometry Analysis of Intracellular Protein 427

nanometer-scale semiconductor particles with unique fluorescence properties (eBioscience ,

The world leader in bringing innovative diagnostic and research tools to different specialists are Beckman Coulter and eBioscience as the major companies producing unconjugated and conjugated antibodies for flow cytometry. BD Biosciences offers a number of Alexa Flouro (AF®) 488/647/700-, APC-, FITC-, PE-, and Pacific BlueTM- conjugated antibodies of BD PharmingenTM and BD PhosflowTM brands used for multicolor flow cytometry (BD

A particularly strong area of leukemia immunophenotyping that contains a broad panel of research products has been built up by (DAKO, 2010). DAKO offers various types of reagents for use in flow cytometry, including primary single-color antibodies conjugated with a single fluorochrome; MultiMix™ Dual-Color Reagents based on the combination of two or more antibodies labeled with FITC and RPE; and MultiMix™ Triple-Color Reagents based on the combination of three antibodies labeled with fluorescein isothiocyanate (FITC), R-phycoerythrin (RPE) and allophycocyanin (APC) or FITC, RPE and RPE-Cy5. Other products for use in flow cytometry are isotype reagents, secondary antibody conjugates, streptavidine conjugates and other accessories. There are also several kits available, e.g. an apoptosis kit used for flow cytometric distinction between viable cells in single cell suspensions or an Enumeration of Stem Cells Kit used for optimal enumeration of CD34+

Another strong company in the production of high-quality activation-state antibodies is Cell Signaling Technology. They offer a number of primary and conjugated antibodies as well as antibody-related kits. Conjugated antibodies are conjugated with AF®, PE or biotin (Cell

There are many other companies not mentioned in the table, including R&D Systems, Miltenyi Biotec and BioLegend, that offer a wide range of unconjugated and conjugated monoclonal antibodies and kits. For example, R&D Systems offers a wide range of biotin-, fluorescein-, PE-, PerCP-, AF® 488-, or APC- conjugated monoclonal antibodies specifically

Generally, the problem with labeling various molecules for flow cytometry in animal species other than mice is the poor availability of directly conjugated primary antibodies. Therefore, indirect labeling is commonly used. This includes labeling cells with a non-conjugated antibody and subsequent visualization with a secondary fluorochrome-conjugated antibody. This indirect labeling is limited by the subclasses of primary antibodies, which should not share the same subclass. This limits the use of the antibodies, especially in the case of

When intracellular labeling is combined with cell surface marker labeling in pigs, the problem with sharing the same subclasses is more noticeable since all the anti-porcine cytokine antibodies used share the most common mouse IgG1 subclass. The cell surface molecules are always labeled prior to the labeling of the intracellular cytokine in our protocol. Accordingly, the anti-cytokine antibody must be directly conjugated with fluorochrome (such as anti-TNF-α or anti-IFN-γ) or it must be biotinylated (such as anti-IL-2

designed to monitor protein expression by flow cytometry (R&D Systems, 2011).

or anti-IL-10) or pre-labeled in some other way (Zelnickova et al., 2008).

Molecular Probes®)..

Biosciences, 2011).

hematopoietic stem /progenitor cells (DAKO, 2010).

Signaling Technology, 2011)

multicolor labeling.

antibodies. Isotype control antibodies are commercially available for both direct and indirect immunofluorescence in the form of fluorochrome-conjugated antibodies and unconjugated antibodies, respectively. During the flow cytometry analysis, the idiotype control antibody is diluted to the same concentration as the specific primary antibody, and is used to stain the sample of negative control cells. This negative control serves to determine the amount of non-specific "background" fluorescence. It allows for setting a threshold of negativity of stained cells. Any event generating a signal above this baseline is considered to be specifically labeled with the target primary antibody.

The isotype control plays an important role during the processing of final measurements. The level of monitored protein is determined as the geometric mean of fluorescent intensity (MFI) of labeled sample, minus the isotype control (O'Gorman et al., 1999, Holden et al., 2006,Hulspas et al., 2009).

Fig. 2. Representative FACS plot showing isotype and CrkL MFI peaks visualized by a FITCconjugated secondary antibody in CD34+ cells isolated from peripheral blood of a newly diagnosed CML patient. The MFI peak of isotype indicates the FITC unspecific background fluorescence. These were visualized using FlowJo software

#### **4. Intracellular antigens for flow cytometry**

The most common way to visualize of the complex between a monoclonal antibody and an antigen in flow cytometry is to covalently bind the antibody to different fluorescent molecules (fluorophores). After exposure to radiation from an excitation source, these fluorophores emit photons with longer wavelengths. Currently, there is a wide range of commercially available fluorophores starting from the small polycyclic molecules, such as fluorescein isothiocyanate (FITC), cyanines, and dyes of the Alexa series, through fluorescent phycobilipoproteins whose best-known representatives are phycoerythrin (PE), allophycocyanin (APC). New way in flow cytometry is also Qdot® nanocrystals –

antibodies. Isotype control antibodies are commercially available for both direct and indirect immunofluorescence in the form of fluorochrome-conjugated antibodies and unconjugated antibodies, respectively. During the flow cytometry analysis, the idiotype control antibody is diluted to the same concentration as the specific primary antibody, and is used to stain the sample of negative control cells. This negative control serves to determine the amount of non-specific "background" fluorescence. It allows for setting a threshold of negativity of stained cells. Any event generating a signal above this baseline is considered to be

The isotype control plays an important role during the processing of final measurements. The level of monitored protein is determined as the geometric mean of fluorescent intensity (MFI) of labeled sample, minus the isotype control (O'Gorman et al., 1999, Holden et al.,

Fig. 2. Representative FACS plot showing isotype and CrkL MFI peaks visualized by a FITCconjugated secondary antibody in CD34+ cells isolated from peripheral blood of a newly diagnosed CML patient. The MFI peak of isotype indicates the FITC unspecific background

The most common way to visualize of the complex between a monoclonal antibody and an antigen in flow cytometry is to covalently bind the antibody to different fluorescent molecules (fluorophores). After exposure to radiation from an excitation source, these fluorophores emit photons with longer wavelengths. Currently, there is a wide range of commercially available fluorophores starting from the small polycyclic molecules, such as fluorescein isothiocyanate (FITC), cyanines, and dyes of the Alexa series, through fluorescent phycobilipoproteins whose best-known representatives are phycoerythrin (PE), allophycocyanin (APC). New way in flow cytometry is also Qdot® nanocrystals –

fluorescence. These were visualized using FlowJo software

**4. Intracellular antigens for flow cytometry** 

specifically labeled with the target primary antibody.

2006,Hulspas et al., 2009).

nanometer-scale semiconductor particles with unique fluorescence properties (eBioscience , Molecular Probes®)..

The world leader in bringing innovative diagnostic and research tools to different specialists are Beckman Coulter and eBioscience as the major companies producing unconjugated and conjugated antibodies for flow cytometry. BD Biosciences offers a number of Alexa Flouro (AF®) 488/647/700-, APC-, FITC-, PE-, and Pacific BlueTM- conjugated antibodies of BD PharmingenTM and BD PhosflowTM brands used for multicolor flow cytometry (BD Biosciences, 2011).

A particularly strong area of leukemia immunophenotyping that contains a broad panel of research products has been built up by (DAKO, 2010). DAKO offers various types of reagents for use in flow cytometry, including primary single-color antibodies conjugated with a single fluorochrome; MultiMix™ Dual-Color Reagents based on the combination of two or more antibodies labeled with FITC and RPE; and MultiMix™ Triple-Color Reagents based on the combination of three antibodies labeled with fluorescein isothiocyanate (FITC), R-phycoerythrin (RPE) and allophycocyanin (APC) or FITC, RPE and RPE-Cy5. Other products for use in flow cytometry are isotype reagents, secondary antibody conjugates, streptavidine conjugates and other accessories. There are also several kits available, e.g. an apoptosis kit used for flow cytometric distinction between viable cells in single cell suspensions or an Enumeration of Stem Cells Kit used for optimal enumeration of CD34+ hematopoietic stem /progenitor cells (DAKO, 2010).

Another strong company in the production of high-quality activation-state antibodies is Cell Signaling Technology. They offer a number of primary and conjugated antibodies as well as antibody-related kits. Conjugated antibodies are conjugated with AF®, PE or biotin (Cell Signaling Technology, 2011)

There are many other companies not mentioned in the table, including R&D Systems, Miltenyi Biotec and BioLegend, that offer a wide range of unconjugated and conjugated monoclonal antibodies and kits. For example, R&D Systems offers a wide range of biotin-, fluorescein-, PE-, PerCP-, AF® 488-, or APC- conjugated monoclonal antibodies specifically designed to monitor protein expression by flow cytometry (R&D Systems, 2011).

Generally, the problem with labeling various molecules for flow cytometry in animal species other than mice is the poor availability of directly conjugated primary antibodies. Therefore, indirect labeling is commonly used. This includes labeling cells with a non-conjugated antibody and subsequent visualization with a secondary fluorochrome-conjugated antibody. This indirect labeling is limited by the subclasses of primary antibodies, which should not share the same subclass. This limits the use of the antibodies, especially in the case of multicolor labeling.

When intracellular labeling is combined with cell surface marker labeling in pigs, the problem with sharing the same subclasses is more noticeable since all the anti-porcine cytokine antibodies used share the most common mouse IgG1 subclass. The cell surface molecules are always labeled prior to the labeling of the intracellular cytokine in our protocol. Accordingly, the anti-cytokine antibody must be directly conjugated with fluorochrome (such as anti-TNF-α or anti-IFN-γ) or it must be biotinylated (such as anti-IL-2 or anti-IL-10) or pre-labeled in some other way (Zelnickova et al., 2008).

Flow Cytometry Analysis of Intracellular Protein 429

Stat5 mAb 47 Ms IgG1 Hu AF-conjugated BD Biosciences Krutzik

Stat6 mAb 18 Ms IgG2a Hu PE-conjugated BD Biosciences Krutzik

CD79a mAb HM57 Ms IgG1 Hu PE-conugated Dako Gerner

Hu – human; Ms – mouse; Mk – monkey; Hm – hamster; Dm – drosophila melanogaster; Bov – bovine;

A method for the direct labeling of antibodies that share the same subclass was tested. Because it was necessary to label relatively small amounts of antibodies, the Zenon-labeling

The Zenon reagents are provided by Invitrogen. This labeling involves the binding of Fab fragments of the fluorochrome-labeled, subclass-specific secondary antibody to the primary antibody, prior to the labeling of the cells. The excess of the fluorochrome-labeled secondary antibody is neutralized by addition of irrelevant mouse antibody in excess, which is supplied within the Zenon kit. The disadvantage of these reagents is the relatively high price. The price is the same for all fluorochromes; however, the number of reactions that can be performed by the kit differ among fluorochromes. Packages with Alexa Fluor, FITC, Texas Red and Pacific dyes contain reagents for 50 rounds of labeling, packages with phycobiliprotein dyes such as R-PE or APC contain reagents for 25 rounds of labeling, and packages with tandem dyes contain reagents for only 10 rounds of labeling. Therefore, it is

The other disadvantage of the Zenon labeling is that the fluorescence yield of Zenon-labeled antibodies is slightly lower compared to classical indirect labeling. Therefore, antibodies against strongly expressed markers should be preferably stained with the Zenon reagents.

1. Place the samples of cell suspensions into a U-bottom 96-well plate, spin the plate,

2. Add the viability staining dye, incubate according the manufacturer's instructions,

3. Add the cocktail of primary antibodies against cell surface molecules in a total volume of 10 μl (dilute antibodies in CWS) + 10 μl of heat-inactivated, filtered goat serum,

4. Add a secondary antibody cocktail in total volume of 25 μl (dilute antibodies in CWS),

If the cell surface molecules are to be labeled with Zenon reagents or if intracellular

If viability staining with permanent dye combined with intracellular staining is

mTOR mAb D9C2 Rab IgG Hu, Ms, Mk, (Rat) unconjugated Cell Signaling

advantageous to choose Zenon reagents containing Alexa Fluor dyes.

General protocol for cell surface staining followed by intracellular labeling

remove as much supernatant as possible, and vortex the plate.

vortex the plate, incubate for 15 min at 4°C, and wash twice.

**Isotype Reactivity Labelling Source Ref.** 

et al., 2003

et al., 2003

et al., 2008

Kalaitzidis et al., 2008

Technology

**Intracellular marker** 

technology was chosen.

(Ondrackova et al., 2010, 2011)

performed, then:

staining follows, then:

vortex the plate, and wash once.

The cell surface staining is performed as follows:

vortex the plate, and incubate for 20 min at 4°C.

**Type Clone name** 

Sc – saccharomyces cerevisiae; Mk – mink; Qua - quail Table 1. Intracellular antigens for flow cytometry


Table 1. continues on next page

Ms IgG1,κ Hu FITC/

IFN- γ mAb P2G10 Ms IgG1,κ Pig PE-conjugate BD Biosciences Gerner

p53 mAb DO7 Ms IgG2b Hu nonconjugated Dako Millard

Rat, Hu

MDR1 mAb JSB-1 Ms IgG1 Hu, Rat, Ms nonconjugated Millipore Millard

PCNA mAb PC10 Ms IgG2a Hu nonconjugated Dako Millard

caspases pAb - Rab IgG Hu, Rat, Ms PE-conjugated BD Biosciences Belloc

perforin mAb dG9 Ms IgG2b Hu PE-conjugated BD Biosciences Gerner

HIV mAb unknown MsIgG1b12 Hu unknown Denis Burton Mascola

*(various) (various) (various) (various)* Afinity

Hu, Ms, Rat, Hm Hu, Ms, Rat, Hm, Mk, Chick, Dm, Bov, Pig, Dog

Sc

ERK1/2 mAb 20A Ms IgG1 Hu, Ms, Rat AF-conjugated BD Biosciences Krutzik

JNK mAb 41 Ms IgG1 Hu unconjugated BD Biosciences Krutzik

p38 MAPK mAb 30 Ms IgG1 Hu, Rat, Ms unconjugated BD Biosciences Krutzik

Stat1 mAb 14 Ms IgG1,κ Hu, Ms unconjugated BD Biosciences Krutzik

Stat4 pAb - Rab IgG Hu, Ms unconjugated Invitrogen Uzel

Stat3 mAb D3A7 Rab IgG Hu, Ms, Rat, Mk AF-conjugated Cell Signaling

mAb 1D5 Ms IgG1 Hu unconjugated Dako Cao

Bax, Bcl-xL, Mcl-1 pAb - Rab IgG Ms/ Rab/ Hu unconjugated Santa Cruz

**Isotype Reactivity Labelling Source Ref.** 

PE/APC/…

conjugated

conjugated

Ms IgG1,κ Hu FITC, PE BD Biosciences Ruitenberg

BD Biosciences Karanikas

Dako Millard

BD Biosciences Gerner

Biotechnology

bioreagents

Technology

Technology

Technology

unconjugated Cell Signaling

unconjugated Cell Signaling

nonconjugated BD Biosciences Millard

et al., 2000

et al., 2008

et al., 1998

et al., 1998

et al., 1998

et al., 1998

et al., 1998

et al., 2000

et al., 2008

et al., 2008

et al., 2002

et al., 2003

et al., 2000

Tazzari et al., 2002

Chow et al., 2001

et al., 2003

et al., 2003

et al., 2003

et al., 2003

Kalaitzidis et al., 2008

et al., 2001

Butts et al., 2007

van Stijn et al., 2003

**Intracellular marker** 

*Cytokines*  IL-2, IL-4, TNF- α, etc.

*Cell cycle or Apoptosis*

*Viral particles* 

*Receptors or their proteins, enzymes* 

Steroid hormone receptor proteins

*Phospho-proteins*  Akt mAb

Estrogen Receptor α Cox1/2 mAb AS70,

mAb pAb

pAb

Table 1. continues on next page

AS67

5G3 -

MEK1/2 pAb - Rab IgG Hu, Ms, Rat, Mk,

**Type Clone name** 

mAb 5344.111, 3010.211, 6401.1111, …

bcl-2 mAb 124 Ms IgG1 Hu FITC-

Ki67 mAb B56 Ms IgG1,κ Ms FITC-

Ms IgG1 Rab IgG

rb mAb G3-245 Ms IgG1 Ms, Qua, Mn, Mk,


Hu – human; Ms – mouse; Mk – monkey; Hm – hamster; Dm – drosophila melanogaster; Bov – bovine; Sc – saccharomyces cerevisiae; Mk – mink; Qua - quail

Table 1. Intracellular antigens for flow cytometry

A method for the direct labeling of antibodies that share the same subclass was tested. Because it was necessary to label relatively small amounts of antibodies, the Zenon-labeling technology was chosen.

The Zenon reagents are provided by Invitrogen. This labeling involves the binding of Fab fragments of the fluorochrome-labeled, subclass-specific secondary antibody to the primary antibody, prior to the labeling of the cells. The excess of the fluorochrome-labeled secondary antibody is neutralized by addition of irrelevant mouse antibody in excess, which is supplied within the Zenon kit. The disadvantage of these reagents is the relatively high price. The price is the same for all fluorochromes; however, the number of reactions that can be performed by the kit differ among fluorochromes. Packages with Alexa Fluor, FITC, Texas Red and Pacific dyes contain reagents for 50 rounds of labeling, packages with phycobiliprotein dyes such as R-PE or APC contain reagents for 25 rounds of labeling, and packages with tandem dyes contain reagents for only 10 rounds of labeling. Therefore, it is advantageous to choose Zenon reagents containing Alexa Fluor dyes.

The other disadvantage of the Zenon labeling is that the fluorescence yield of Zenon-labeled antibodies is slightly lower compared to classical indirect labeling. Therefore, antibodies against strongly expressed markers should be preferably stained with the Zenon reagents. (Ondrackova et al., 2010, 2011)

General protocol for cell surface staining followed by intracellular labeling


The cell surface staining is performed as follows:


Flow Cytometry Analysis of Intracellular Protein 431

(b) Fig. 3. Seven color flow cytometry for identification of porcine monocyte subpopulations

The red blood cells from the whole peripheral blood or from the bone marrow sample were lysed with ammonium chloride solution. The following fluorescent staining was performed:

Primary antibody Secondary antibody / Zenon reagent / viability stain

VMRD, USA 0.1 μl 3) IgG1 AlexaFluor488 Invitrogen,

1 μl 3) IgM DyLight405 GeneTex,

APC-AlexaFluor750

IgG1 \*\*

IgG1 \*\*\*

Class/ subclass Fluorochrome Manufacturer Dilution Stained

USA

USA

USA

USA

Invitrogen, USA

AlexaFluor647 Invitrogen, USA

at point

1:750 4)

1:750 4)

1:100 4)

1:500 4)

Stained at point

488 Propidium iodide 11)

using two Zenon-labeled antibodies

Clone Manufacturer Amount

Lunney \*

Solution B of the Zenon kit, mix well, incubate 10 min at 4°C

of Solution B of the Zenon kit, mix well, incubate 10 min at 4°C

per well

488 CD14 MIL-2 Serotec, UK 1 μl 3) IgG2b AlexaFluor647 Invitrogen,

488 SLA-DR MSA3 VMRD, USA 0.05 μl 3) IgG2a PE-Cy5.5 Invitrogen,

VMRD, USA 0.1 μl 7) Zenon

Serotec, UK 0.1 μl 7) Zenon

\* a generous gift from Dr. J.K. Lunney, Animal Parasitology Institute, Beltsville, MO, USA

\*\* labeling with the Zenon® AlexaFluor647 Mouse IgG1 Labeling Kit perform as follows: 0.1 μl of anti-CD172α and 0.5 μl of Solution A of the Zenon Kit, mix well, incubate 10 min at 4°C, then add 0.5 μl of

\*\*\* labeling with the Zenon® APC-AlexaFluor750 Mouse IgG1 Labeling Kit perform as follows: 0.1 μl of anti- CD203α and 0.5 μl of Solution A of the Zenon kit, mix well, incubate 10 min at 4°C, then add 0.5 μl

The measurement was performed by using BD FACSAria I flow cytometer (Becton Dickinson, USA).

Excitation

wavelength

Target molecule

488 CD163 2A10/

11

488 SWC8 MIL-3 Dr. J.K.

B

18-7

640 CD172α DH59

640 CD203α PM


CWS solution: PBS containing 1.84 g/l EDTA, 1 g/l NaN3, 4 ml/l gelatin from cold water fish skin

Wash definition: Add as much CWS into each well as possible, spin 3 min at 500 g, remove much supernatant as possible, and vortex.

5. Wash once and vortex the plate thoroughly because the plate in the next step cannot be vortexed due to the risk of the samples overflowing into the neighboring wells. 6. Add 100 μl of heat-inactivated, filtered mouse serum diluted 1:10 in CWS, incubate for

Labeling cell surface molecules with Zenon-labeled antibodies now follows, but this

Staining of intracellular molecules with directly-labeled, Zenon-labeled, or unlabeled

7. Add the cocktail of Zenon-labeled antibodies against cell surface molecules in a total volume of 10 μl, incubate for 15 min at 4°C, vortex the plate, and wash –twice.

8. Add 30 μl of Solution A from the IntraStain kit, vortex thoroughly to allow complete hemolysis of contaminating red blood cells, incubate for 15 min at room temperature,

9. Add primary antibodies against intracellular molecules (directly-labeled, Zenonlabeled, or unlabeled) diluted in Solution B from the IntraStain kit in a total volume of 20 μl, vortex the plate, incubate for 20 min at room temperature, and wash twice. 10. Add secondary antibodies diluted in Solution B from the InraStain kit and CWS (ratio of Solution B and CWS 1:1) in total volume of 25 μl, incubate for 20 min at room

11. Resuspend samples in CWS in the volume that is required for the subsequent

CWS solution: PBS containing 1.84 g/l EDTA, 1 g/l NaN3, 4 ml/l gelatin from cold water

Wash definition: Add as much CWS into each well as possible, spin 3 min at 500 g, remove

(a)

antibodies follows, but these steps can be omitted if they not required:

measurement and measure by the appropriate method.

20 min at 4°C, and wash once.

and wash –twice.

fish skin

temperature, wash twice.

much supernatant as possible, and vortex.

step can be omitted if it is not required:

Fig. 3. Seven color flow cytometry for identification of porcine monocyte subpopulations using two Zenon-labeled antibodies



\* a generous gift from Dr. J.K. Lunney, Animal Parasitology Institute, Beltsville, MO, USA

\*\* labeling with the Zenon® AlexaFluor647 Mouse IgG1 Labeling Kit perform as follows: 0.1 μl of anti-CD172α and 0.5 μl of Solution A of the Zenon Kit, mix well, incubate 10 min at 4°C, then add 0.5 μl of Solution B of the Zenon kit, mix well, incubate 10 min at 4°C

\*\*\* labeling with the Zenon® APC-AlexaFluor750 Mouse IgG1 Labeling Kit perform as follows: 0.1 μl of anti- CD203α and 0.5 μl of Solution A of the Zenon kit, mix well, incubate 10 min at 4°C, then add 0.5 μl of Solution B of the Zenon kit, mix well, incubate 10 min at 4°C

The measurement was performed by using BD FACSAria I flow cytometer (Becton Dickinson, USA).

Flow Cytometry Analysis of Intracellular Protein 433

Class/subclass Fluorochrome Manufacturer Dilution Stained

Western FACS

at point

Primary antibody Secondary antibody / Zenon reagent / viability stain

405 SWC8 MIL-3 Dr. J.K. Lunney \* 1 μl 3) IgM DyLight405 GeneTex, USA 1:500 4) 405 LIVEDEAD® Fixable Aqua Dead Cell Stain Kit \*\* 2) 488 CD163 2A10/11 VMRD, USA 0.1 μl 3) IgG1 AlexaFluor488 Invitrogen, USA 1:750 4)

561 SLA-DR MSA3 VMRD, USA 0.05 μl 3) IgG2a PE-Cy5.5 Invitrogen, USA 1:100 4) 640 CD14 MIL-2 Serotec, UK 1 μl 3) IgG2b AlexaFluor647 Invitrogen, USA 1:750 4)

\*\*\* labeling with the Zenon® R-Phycoerythrin Mouse IgG1 Labeling Kit perform as follows: 0.2 μl of anti-IL-8 and 4 μl of Solution A of the Zenon kit, mix well, incubate 10 min at 4°C, then add 4 μl of solution B of the Zenon kit, mix well, incubate 10 min at 4°C, then add 11.8 μl of Solution B of IntraStain

The measurement was performed by using BD LSRFortessa flow cytometer (Becton Dickinson, USA)

Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cells that is characterized by the presence of the BCR-ABL fusion gene, which encodes the constitutively active BCR-ABL tyrosine kinase (Daley et al., 1990). Currently, the tyrosine kinase inhibitor imatinib (IM) (a potent inhibitor of BCR-ABL) is used as a first line therapy

0 0.1 0.5 1 5 10

Fig. 5. The equivalence between flow cytometry and Western blot methods, in p-CrkL reduction in a BCR-ABL positive K562 cell line after 48 h treatment with imatinib (Hamilton

IM concentration (μM)

**5. Intracellular measurement of the p-CrkL and CrkL levels using flow** 

Stained at point

561 IL-8 8M6 Serotec, UK 0.2 μl 9) Zenon IgG1 \*\*\* R-PE Invitrogen, USA

\* a generous gift from Dr. J.K. Lunney, Animal Parasitology Institute, Beltsville, MO, USA

Excitation

kit

**cytometry** 

**5.1 Theoretical background** 

100

80

60

40

% P-CrkL

20

0

et al., 2006)

for CML patients (Baccarani et al., 2009).

wavelength

Target molecule Clone Manufacturer Amount

\*\* diluted 1:1000 in PBS, 10 μl / well, incubation15 min at 4°C

per well

The gating strategy for identification of monocytes in the bone marrow is depicted (A). Briefly, the leukocytes were gated according their light scatter properties (upper left dotplot). The dublets of cells were excluded from the further analysis (upper middle dot-plot). The viable (propidium iodide-negative) cell were gated (upper right dot-plot). The CD203αpositive macrophages were excluded (lower left dot-plot). The monocytes were identified as SWC8-negative (lower middle dot-plot) CD172α-positive cells (lower right dot-plot).

Then monocyte subpopulations from the bone marrow and peripheral blood were identified based on expression of SLA-DR, CD14 and CD163 (B). The SLA-DR-positive and negative monocytes were gated (left dot-plots). Then SLA-DR-negative (middle dot-plots) and SLA-DR-positive (right dot-plots) monocyte subpopulations were depicted in CD163 vs. CD14 dot-plots.

Fig. 4. Six color flow cytometry for measurement of IL-8 production by monocyte subpopulations using the Zenon-labeled anti-IL-8 antibody

The gating order for evaluation of IL-8 production by monocyte subpopulations is depicted (A). Briefly, the leukocytes were gated according their light scatter properties (B). The dublets of cells were excluded excluded from the further analysis (C). The viable (LIVEDEAD® Fixable Aqua Dead Cell Stain-negative) cell were gated (D). The monocytes were identified as SWC8-negative CD14-positive cells (E). The IL-8 production by CD163 positive and negative monocytes (F) and by SLA-DR-positive and negative monocytes (G) was then evaluated.

The whole peripheral blood diluted 1:1 with RPMI 1640 was stimulated for 2 hours with LPS (1 μg/ml) in the presence of brefeldin A (10 μg/ml). The red blood cells were lysed with ammonium chloride solution. The following fluorescent staining was performed:

The gating strategy for identification of monocytes in the bone marrow is depicted (A). Briefly, the leukocytes were gated according their light scatter properties (upper left dotplot). The dublets of cells were excluded from the further analysis (upper middle dot-plot). The viable (propidium iodide-negative) cell were gated (upper right dot-plot). The CD203αpositive macrophages were excluded (lower left dot-plot). The monocytes were identified as

Then monocyte subpopulations from the bone marrow and peripheral blood were identified based on expression of SLA-DR, CD14 and CD163 (B). The SLA-DR-positive and negative monocytes were gated (left dot-plots). Then SLA-DR-negative (middle dot-plots) and SLA-DR-positive (right dot-plots) monocyte subpopulations were depicted in CD163 vs. CD14

SWC8-negative (lower middle dot-plot) CD172α-positive cells (lower right dot-plot).

Fig. 4. Six color flow cytometry for measurement of IL-8 production by monocyte

The gating order for evaluation of IL-8 production by monocyte subpopulations is depicted (A). Briefly, the leukocytes were gated according their light scatter properties (B). The dublets of cells were excluded excluded from the further analysis (C). The viable (LIVEDEAD® Fixable Aqua Dead Cell Stain-negative) cell were gated (D). The monocytes were identified as SWC8-negative CD14-positive cells (E). The IL-8 production by CD163 positive and negative monocytes (F) and by SLA-DR-positive and negative monocytes (G)

The whole peripheral blood diluted 1:1 with RPMI 1640 was stimulated for 2 hours with LPS (1 μg/ml) in the presence of brefeldin A (10 μg/ml). The red blood cells were lysed with ammonium chloride solution. The following fluorescent staining was performed:

subpopulations using the Zenon-labeled anti-IL-8 antibody

dot-plots.

was then evaluated.


\* a generous gift from Dr. J.K. Lunney, Animal Parasitology Institute, Beltsville, MO, USA \*\* diluted 1:1000 in PBS, 10 μl / well, incubation15 min at 4°C

\*\*\* labeling with the Zenon® R-Phycoerythrin Mouse IgG1 Labeling Kit perform as follows: 0.2 μl of anti-IL-8 and 4 μl of Solution A of the Zenon kit, mix well, incubate 10 min at 4°C, then add 4 μl of solution B of the Zenon kit, mix well, incubate 10 min at 4°C, then add 11.8 μl of Solution B of IntraStain kit

The measurement was performed by using BD LSRFortessa flow cytometer (Becton Dickinson, USA)

#### **5. Intracellular measurement of the p-CrkL and CrkL levels using flow cytometry**

#### **5.1 Theoretical background**

Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cells that is characterized by the presence of the BCR-ABL fusion gene, which encodes the constitutively active BCR-ABL tyrosine kinase (Daley et al., 1990). Currently, the tyrosine kinase inhibitor imatinib (IM) (a potent inhibitor of BCR-ABL) is used as a first line therapy for CML patients (Baccarani et al., 2009).

Fig. 5. The equivalence between flow cytometry and Western blot methods, in p-CrkL reduction in a BCR-ABL positive K562 cell line after 48 h treatment with imatinib (Hamilton et al., 2006)

Flow Cytometry Analysis of Intracellular Protein 435

Sample Name

**IC50imatinib**

**p-CrkL ratio**

CrkL untreated.fcs p-CrkL 5μM IM.fcs p-CrkL 1.5μM IM.fcs p-CrkL 0.5μM IM.fcs p-CrkL untreated.fcs

Isotype.fcs

0 10 10 10 10 2 3 4 5 Comp-FITC-A

Fig. 6. Representative FACS plot showing isotype, p-CrkL and CrkL MFI peaks in CD34+ cells isolated from peripheral blood of a newly diagnosed CML patient. Changes in p-CrkL MFI peaks following *in vitro* imatinib (IM) treatment are detectable and were visualized

> 0 1 2 3 4 5 imatinib concentration (μM)

This work was generously supported by grant of the Ministry of Education of the Czech Republic MSM 0021622430, grant and Ministryof Health NS-9681 and Ministry of

Fig. 7. The graph of p-CrkL decrease upon *in vitro* imatinib treatment. MFI peaks

quantification was calculated in FlowJo software

Agriculture of the Czech Republic MZE0002716202

Count

0

using FlowJo software

100

80

60

p-CrkL ratio (%)

40

20

0

**6. Acknowledgment** 

The CrkL protein is a downstream signaling substrate of BCR-ABL, and its tyrosine phosphorylation (p-CrkL) serves as a specific indicator of BCR-ABL kinase activity in CML cells (Nichols et al., 1994; Patel et al., 2006). Recent studies have revealed that p-CrkL can act as a prognostic marker for imatinib treatment response of CML patients using either western blotting (White et al., 2005) or flow cytometry (Lucas et al., 2010). However, certain discrepancies have been found in the literature concerning the predictive value of p-CrkL in different cell types (mononuclear cells or CD34+ cells) used for analysis (Khorashad et al., 2009).

The technique for measuring p-CrkL levels using flow cytometry was originally described by Hamilton et al., (Hamilton et al., 2006) and the equivalence between flow cytometry and western blot methods was demonstrated (Figure 5).

#### **5.2 The technique of intracellular p-CrkL and CrkL measurement by flow cytometry**

Mononuclear cells (MNCs) were isolated from PB of newly diagnosed CML patients using Histopaque-1077 density gradient centrifugation (Sigma–Aldrich, St. Louis, MO, USA) and subsequently enriched for CD34+ cells using magnetic-activated cell sorting (MACS) with a CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. CD34+ cells (5x104) were incubated for 16 h with 0, 0.5, 1.5, and 5 μM imatinib in 1 ml of serum-free medium (SFEM), supplemented with StemSpan CC100 cytokine mixture (StemCell Technologies, Köln, Germany) as previously described (Koutna et al., 2011). Then the cells were washed in phosphate buffered saline (PBS) and fixed with 4% formaldehyde for 10 min at 37°C, then washed in PBS and permeabilized by 90% methanol at 4°C for 30 min.

After permeabilization, the cells were washed in PBS and incubated with primary unlabeled antibody for 30 min at 4°C in 100 μl of FACS incubation buffer (0.5% bovine serum albumin in PBS). The concentration of primary antibodies was 12 µg/ml (p-CrkL, Cell Signaling Technology, Danvers, MA, USA; CrkL, Santa Cruz biotechnology, Santa Cruz, CA, USA; isotype control anti-normal-rabbit IgG G, R&D Systems, Minneapolis, MN, USA). The cells were washed in FACS incubation buffer and incubated with FITC-conjugated anti-rabbit IgG secondary antibody (Sigma-Aldrich) in a concentration of 10 µl/ml.

All samples were measured on a FACSCanto II Flow Cytometer (Becton Dickinson). For data analysis, BD FACSDiva (Becton-Dickinson) and FlowJo (Tree Star, Ashland, USA) software were used. The viable cell population was gated according to forward scatter and side scatter parameters. The level of p-CrkL and CrkL in the viable cells was determined as the geometric mean fluorescence intensity (MFI) of the p-CrkL- or CrkL-labeled sample minus the MFI of the isotype control (Figure 6).

The IC50imatinib was defined as the concentration of imatinib that caused a 50% decrease in the amount of p-CrkL compared to the untreated control (Figure 7) (White et al., 2005). The p-CrkL/CrkL ratio was calculated by dividing the concentrations of p-CrkL by those of CrkL and multiplying by 100 in untreated cells (Lucas et al., 2010). The p-CrkL ratio was assessed as a percentage of p-CrkL in the samples treated with a maximal imatinib concentration (5 µM) relative to the untreated control (Figure 7) (Khorashad et al., 2009).

The CrkL protein is a downstream signaling substrate of BCR-ABL, and its tyrosine phosphorylation (p-CrkL) serves as a specific indicator of BCR-ABL kinase activity in CML cells (Nichols et al., 1994; Patel et al., 2006). Recent studies have revealed that p-CrkL can act as a prognostic marker for imatinib treatment response of CML patients using either western blotting (White et al., 2005) or flow cytometry (Lucas et al., 2010). However, certain discrepancies have been found in the literature concerning the predictive value of p-CrkL in different cell types (mononuclear cells or CD34+ cells) used for analysis (Khorashad et al.,

The technique for measuring p-CrkL levels using flow cytometry was originally described by Hamilton et al., (Hamilton et al., 2006) and the equivalence between flow cytometry and

**5.2 The technique of intracellular p-CrkL and CrkL measurement by flow cytometry** 

Mononuclear cells (MNCs) were isolated from PB of newly diagnosed CML patients using Histopaque-1077 density gradient centrifugation (Sigma–Aldrich, St. Louis, MO, USA) and subsequently enriched for CD34+ cells using magnetic-activated cell sorting (MACS) with a CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. CD34+ cells (5x104) were incubated for 16 h with 0, 0.5, 1.5, and 5 μM imatinib in 1 ml of serum-free medium (SFEM), supplemented with StemSpan CC100 cytokine mixture (StemCell Technologies, Köln, Germany) as previously described (Koutna et al., 2011). Then the cells were washed in phosphate buffered saline (PBS) and fixed with 4% formaldehyde for 10 min at 37°C, then washed in PBS and permeabilized by 90%

After permeabilization, the cells were washed in PBS and incubated with primary unlabeled antibody for 30 min at 4°C in 100 μl of FACS incubation buffer (0.5% bovine serum albumin in PBS). The concentration of primary antibodies was 12 µg/ml (p-CrkL, Cell Signaling Technology, Danvers, MA, USA; CrkL, Santa Cruz biotechnology, Santa Cruz, CA, USA; isotype control anti-normal-rabbit IgG G, R&D Systems, Minneapolis, MN, USA). The cells were washed in FACS incubation buffer and incubated with FITC-conjugated anti-rabbit

All samples were measured on a FACSCanto II Flow Cytometer (Becton Dickinson). For data analysis, BD FACSDiva (Becton-Dickinson) and FlowJo (Tree Star, Ashland, USA) software were used. The viable cell population was gated according to forward scatter and side scatter parameters. The level of p-CrkL and CrkL in the viable cells was determined as the geometric mean fluorescence intensity (MFI) of the p-CrkL- or CrkL-labeled sample

The IC50imatinib was defined as the concentration of imatinib that caused a 50% decrease in the amount of p-CrkL compared to the untreated control (Figure 7) (White et al., 2005). The p-CrkL/CrkL ratio was calculated by dividing the concentrations of p-CrkL by those of CrkL and multiplying by 100 in untreated cells (Lucas et al., 2010). The p-CrkL ratio was assessed as a percentage of p-CrkL in the samples treated with a maximal imatinib concentration (5 µM) relative to the untreated control (Figure 7) (Khorashad et al., 2009).

IgG secondary antibody (Sigma-Aldrich) in a concentration of 10 µl/ml.

minus the MFI of the isotype control (Figure 6).

western blot methods was demonstrated (Figure 5).

methanol at 4°C for 30 min.

2009).

Fig. 6. Representative FACS plot showing isotype, p-CrkL and CrkL MFI peaks in CD34+ cells isolated from peripheral blood of a newly diagnosed CML patient. Changes in p-CrkL MFI peaks following *in vitro* imatinib (IM) treatment are detectable and were visualized using FlowJo software

Fig. 7. The graph of p-CrkL decrease upon *in vitro* imatinib treatment. MFI peaks quantification was calculated in FlowJo software

#### **6. Acknowledgment**

This work was generously supported by grant of the Ministry of Education of the Czech Republic MSM 0021622430, grant and Ministryof Health NS-9681 and Ministry of Agriculture of the Czech Republic MZE0002716202

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**22** 

*Brazil* 

Silvana Gaiba et al\*,

**Biological Effects Induced by** 

*Universidade Federal de São Paulo – Unifesp, Universidade Estadual de Santa Cruz – Uesc Universidade Nove de Julho – Uninove* 

**Ultraviolet Radiation in Human Fibroblasts** 

As the most superficial body organ, skin plays an important role in protecting the body from environmental damage. The skin is composed of three layers: the epidermis, dermis and subcutaneous tissue. The epidermis, the outermost layer, has as main functions to protect the body against harmful environmental stimuli and to reduce fluid loss. It is a stratified squamous epithelium with several layers and its major cell type is the keratinocyte. This tissue is constantly being renewed by keratinization, a process of detachment of cornified cells (Blumenberg & Tomic-Canic, 1997). Located under the epidermis are the dermis and the dermal connective tissue, with extracellular matrix proteins such as collagen, elastic fibers, fibronectin, glycosaminoglycans and proteoglycans, which are produced and secreted into the extracellular space by fibroblasts, the major cell type found in this tissue (Makrantonaki & Zouboulis, 2007). The extracellular matrix proteins in the dermal connective tissue contribute for maintaining skin preservation and integrity (Hwang *et al*., 2011). Stromal fibroblasts play an important role in tissue homeostasis regulation and wound repair via protein synthesis and secretion of growth factors or cytokines of paracrine action with direct effect on proliferation and differentiation of adjacent epithelial tissues (Andriani *et al*., 2011). Solar ultraviolet (UV) radiation is a predictable epidemiologic risk factor for melanoma and non-melanoma skin cancers. (Katiyar *et al*., 2011). UV irradiation can impair cellular functions by directly damaging DNA to induce apoptosis (Wäster & Ollinger, 2009). Among other things, longer UV wavelengths (UVB, UVA) induce oxidative stress and protein denaturation whereas short wavelength UV radiation (UVC) causes predominantly DNA damage to cells in the form of pyrimidine dimers, 6-4 photoproducts and apoptosis (Armstrong & Kricker, 2001; Gruijl *et al*., 2001). UVB irradiation damages skin cells by the formation of ROS (Reactive Oxygen Species) resulting in oxidative stress, an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA (Wäster & Ollinger, 2009). However, it has less penetrating power than UVA and acts mainly on the epidermal basal layer of the skin. UVC, on the other hand, is extremely damaging to the skin because its wavelengths have enormous energy and induce genotoxic

Vanina M. Tucci-Viegas, Lucimar P. França, Fernanda Lasakosvitsch, Fernanda L. A. Azevedo, Andrea

A. F. S. Moraes, Alice T. Ferreira and Jerônimo P. França

**1. Introduction** 

 \*

apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia. Leukemia, 17, 4, 780-6, 0887-6924


BD Biosciences; http://www.bdbiosciences.com

Beckman Coulter https://www.beckmancoulter.com

Cell Signaling Technology; http://www.cellsignal.com

Dako; http://www.dako.com

eBioscience (2011) http://www.ebioscience.com

R&D Systems http://www.rndsystems.com

### **Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts**

#### Silvana Gaiba et al\*,

*Universidade Federal de São Paulo – Unifesp, Universidade Estadual de Santa Cruz – Uesc Universidade Nove de Julho – Uninove Brazil* 

#### **1. Introduction**

438 Flow Cytometry – Recent Perspectives

White, D., Saunders, V., Lyons, A., Branford, S., Grigg, A., To, L., & Hughes, T. (2005). In

Zelnickova, P., Faldyna, M., Stepanova, H., Ondracek, J., & Kovaru, F. (2007). Intracellular

Zelnickova, P., Leva, L., Stepanova, H., Kovaru, F., & Faldyna, M. (2008). Age-dependent

phagocytes. Vet Immunol Immunopathol, 124, 3-4, 367-78, 0165-2427

surface staining. J Immunol Methods, 327, 1-2, 18-29, 0022-1759

leukemia. Leukemia, 17, 4, 780-6, 0887-6924

BD Biosciences; http://www.bdbiosciences.com Beckman Coulter https://www.beckmancoulter.com Cell Signaling Technology; http://www.cellsignal.com

eBioscience (2011) http://www.ebioscience.com R&D Systems http://www.rndsystems.com

4971

Dako; http://www.dako.com

apoptosis-related protein expression in minimal residual disease in acute myeloid

vitro sensitivity to imatinib-induced inhibition of ABL kinase activity is predictive of molecular response in patients with de novo CML. Blood, 106, 7, 2520-6, 0006-

cytokine detection by flow cytometry in pigs: fixation, permeabilization and cell

changes of proinflammatory cytokine production by porcine peripheral blood

As the most superficial body organ, skin plays an important role in protecting the body from environmental damage. The skin is composed of three layers: the epidermis, dermis and subcutaneous tissue. The epidermis, the outermost layer, has as main functions to protect the body against harmful environmental stimuli and to reduce fluid loss. It is a stratified squamous epithelium with several layers and its major cell type is the keratinocyte. This tissue is constantly being renewed by keratinization, a process of detachment of cornified cells (Blumenberg & Tomic-Canic, 1997). Located under the epidermis are the dermis and the dermal connective tissue, with extracellular matrix proteins such as collagen, elastic fibers, fibronectin, glycosaminoglycans and proteoglycans, which are produced and secreted into the extracellular space by fibroblasts, the major cell type found in this tissue (Makrantonaki & Zouboulis, 2007). The extracellular matrix proteins in the dermal connective tissue contribute for maintaining skin preservation and integrity (Hwang *et al*., 2011). Stromal fibroblasts play an important role in tissue homeostasis regulation and wound repair via protein synthesis and secretion of growth factors or cytokines of paracrine action with direct effect on proliferation and differentiation of adjacent epithelial tissues (Andriani *et al*., 2011). Solar ultraviolet (UV) radiation is a predictable epidemiologic risk factor for melanoma and non-melanoma skin cancers. (Katiyar *et al*., 2011). UV irradiation can impair cellular functions by directly damaging DNA to induce apoptosis (Wäster & Ollinger, 2009). Among other things, longer UV wavelengths (UVB, UVA) induce oxidative stress and protein denaturation whereas short wavelength UV radiation (UVC) causes predominantly DNA damage to cells in the form of pyrimidine dimers, 6-4 photoproducts and apoptosis (Armstrong & Kricker, 2001; Gruijl *et al*., 2001). UVB irradiation damages skin cells by the formation of ROS (Reactive Oxygen Species) resulting in oxidative stress, an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA (Wäster & Ollinger, 2009). However, it has less penetrating power than UVA and acts mainly on the epidermal basal layer of the skin. UVC, on the other hand, is extremely damaging to the skin because its wavelengths have enormous energy and induce genotoxic

<sup>\*</sup> Vanina M. Tucci-Viegas, Lucimar P. França, Fernanda Lasakosvitsch, Fernanda L. A. Azevedo, Andrea A. F. S. Moraes, Alice T. Ferreira and Jerônimo P. França

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts 441

activation, an essential outcome of the DNA damage response pathway, leads to cell cycle arrest and DNA repair, apoptosis, or cellular senescence. More specifically, initial genomic insults lead to p53 stabilization and nuclear localization where transient cell cycle arrest can be quickly activated, allowing damaged DNA repair prior to replication. A signaling cascade can be activated by p53 in case of extreme and irreversible DNA damage to induce

UV radiation induces phospholipids peroxidation in cellular membrane. Lipid peroxidation is a consequence of free primary radicals (ROS). This, in turn, leads to the generation of polar products and increase the membrane dielectric constant and capacitance. An important consequence of this phenomenon is the alteration of transport particles across the

During the Fenton reaction, singlet oxygen directly initiates lipid peroxides and hydrogen

Cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence are induced by the p53 tumor suppressor pathway upon activation by genotoxic stress. This pathway works mostly through transactivation of different downstream targets, for example, p21 cell cycle inhibitor, required for short-term cell cycle arrest or long-term cellular senescence, or other proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) (Tavana *et al*., 2010). Yet, the mechanism that regulates the switching from cell cycle arrest to apoptosis is still unknown. In case of extreme or irreversible damage, p53 can additionally activate a signaling cascade to induce apoptosis through transcription of pro-apoptotic genes, most particularly p53-upregulated modulator of apoptosis (PUMA) and trans-repression of anti-apoptotic genes including Bcl-2. Programmed cell death directly protects cells against the accumulation of genomic

Fig. 1. Schematic representation. Photobiological effects of ultraviolet radiation on human

programmed cell death through transcription of different proapoptotic factors.

peroxides indirectly initiate hydroxyl radicals (Halliwell & Gutteridge, 1999).

membrane (Strässle *et al*., 1991)

instability that could lead to tumorigenesis.

skin cells

stress. Fortunately, UVC is prevented from reaching the earth, as it is largely absorbed by atmospheric ozone layer (Afag, 2011). It has already been proposed that programmed cell death (apoptosis) can be induced by UV light in various cell types (reviewed in Schwarz, 1998). The cellular responses to injuries or stresses are important in determining cell fate (Aylon & Oren, 2007). Many signaling pathways participate in this process, with the mitogen-activated protein kinase (MAPK) cascades and p53 pathway being two of the major pathways implicated (Aylon & Oren, 2007; Li *et al*., 2009). The cellular response to DNA damage is focused on p53, which can induce the cell to apoptosis by the protein PUMA (p53 up-regulated modulator of apoptosis), a member of the Bcl-2 homology (BH)3-only Bcl-2 family proteins. Recent studies suggest that Bcl-2 family members play an essential role in regulating apoptosis initiation through the mitochondria (Zhang *et al*., 2009). UV irradiation induces permeabilization of the lysosomal membrane with release of cathepsin B and D to the cytosol, translocation of the proapoptotic Bcl-2 proteins Bax and Bid to mitochondriallike structures. Subsequently, there is cytochrome c release and activation of caspase-3 (Bivik *et al*., 2006). p38 MAPK, one of the four MAPK subfamilies in mammalian cells, is activated by proinammatory cytokines and environmental stress (Brown & Benchimol, 2006; Johnson & Lapadat, 2002). p38 is not only reported to be phosphorylated and activated to mediate cell apoptosis and the differentiation process (Thornton & Rincon, 2009), but also to have cell protective effects under certain circumstances (Chouinard *et al*., 2002). MAPK pathways mediate cellular responses to many different extracellular signaling molecules such as the ones involved in differentiation, gene expression, regulation of proliferation, apoptosis, development, motility or metabolism. The typical MAPK pathways, characterized by the ERK1/2, ERK5, JNK, and p38MAPK components, comprise a cascade of three successive phosphorylation events exerted by a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK), and a MAPK (Kostenko *et al*., 2011).

Ultraviolet UVA light absorption after solar exposure is responsible for photoactivation of DNA and other biomolecules. Additionally, UVA radiation (320-400 nm) induces photoaddition, oxidative stress and DNA damage, which may be continuous. The cell is also unable to replicate in case of severe DNA damage. This way, DNA repair must be considered essential for genetic information preservation and transmission in any life form. UVA light generates mutagenic DNA lesions in the skin. Exposure to solar UVB radiation is responsible for skin inammation and tumorigenesis.

Besides that, oxidative stress induced by solar radiation could be responsible, as well, for the increased frequency of DNA mutations in photoaged human skin. Genomic DNA damage triggers the activation of a network of pathways that rapidly modulate several cellular activities. ROS and hydrogen peroxide can damage DNA. Furthermore, it has been recently shown that increased oxidative stress is correlated to DNA alterations. ROS are deleterious to DNA, membranes and proteins although their exact role in mutagenesis and lethality is still unclear in the many skin cell types. In addition, repair ability and defense mechanisms may differ a lot from one cellular type to another.

Epidermal and dermal cells are targets for UVA oxidative stress and their antioxidant defenses can be defeated. Keratinocytes and fibroblasts may respond differently to UV radiation depending on their localization in the body or their functional and metabolic characteristics. Cell culture models have helped to describe the cytotoxic action of UVA and the role of ROS in UVA-induced cellular damage (Tyrrell, 1990). p53 stabilization and

stress. Fortunately, UVC is prevented from reaching the earth, as it is largely absorbed by atmospheric ozone layer (Afag, 2011). It has already been proposed that programmed cell death (apoptosis) can be induced by UV light in various cell types (reviewed in Schwarz, 1998). The cellular responses to injuries or stresses are important in determining cell fate (Aylon & Oren, 2007). Many signaling pathways participate in this process, with the mitogen-activated protein kinase (MAPK) cascades and p53 pathway being two of the major pathways implicated (Aylon & Oren, 2007; Li *et al*., 2009). The cellular response to DNA damage is focused on p53, which can induce the cell to apoptosis by the protein PUMA (p53 up-regulated modulator of apoptosis), a member of the Bcl-2 homology (BH)3-only Bcl-2 family proteins. Recent studies suggest that Bcl-2 family members play an essential role in regulating apoptosis initiation through the mitochondria (Zhang *et al*., 2009). UV irradiation induces permeabilization of the lysosomal membrane with release of cathepsin B and D to the cytosol, translocation of the proapoptotic Bcl-2 proteins Bax and Bid to mitochondriallike structures. Subsequently, there is cytochrome c release and activation of caspase-3 (Bivik *et al*., 2006). p38 MAPK, one of the four MAPK subfamilies in mammalian cells, is activated by proinammatory cytokines and environmental stress (Brown & Benchimol, 2006; Johnson & Lapadat, 2002). p38 is not only reported to be phosphorylated and activated to mediate cell apoptosis and the differentiation process (Thornton & Rincon, 2009), but also to have cell protective effects under certain circumstances (Chouinard *et al*., 2002). MAPK pathways mediate cellular responses to many different extracellular signaling molecules such as the ones involved in differentiation, gene expression, regulation of proliferation, apoptosis, development, motility or metabolism. The typical MAPK pathways, characterized by the ERK1/2, ERK5, JNK, and p38MAPK components, comprise a cascade of three successive phosphorylation events exerted by a MAPK kinase kinase (MAPKKK), a

Ultraviolet UVA light absorption after solar exposure is responsible for photoactivation of DNA and other biomolecules. Additionally, UVA radiation (320-400 nm) induces photoaddition, oxidative stress and DNA damage, which may be continuous. The cell is also unable to replicate in case of severe DNA damage. This way, DNA repair must be considered essential for genetic information preservation and transmission in any life form. UVA light generates mutagenic DNA lesions in the skin. Exposure to solar UVB radiation is

Besides that, oxidative stress induced by solar radiation could be responsible, as well, for the increased frequency of DNA mutations in photoaged human skin. Genomic DNA damage triggers the activation of a network of pathways that rapidly modulate several cellular activities. ROS and hydrogen peroxide can damage DNA. Furthermore, it has been recently shown that increased oxidative stress is correlated to DNA alterations. ROS are deleterious to DNA, membranes and proteins although their exact role in mutagenesis and lethality is still unclear in the many skin cell types. In addition, repair ability and defense mechanisms

Epidermal and dermal cells are targets for UVA oxidative stress and their antioxidant defenses can be defeated. Keratinocytes and fibroblasts may respond differently to UV radiation depending on their localization in the body or their functional and metabolic characteristics. Cell culture models have helped to describe the cytotoxic action of UVA and the role of ROS in UVA-induced cellular damage (Tyrrell, 1990). p53 stabilization and

MAPK kinase (MAPKK), and a MAPK (Kostenko *et al*., 2011).

responsible for skin inammation and tumorigenesis.

may differ a lot from one cellular type to another.

activation, an essential outcome of the DNA damage response pathway, leads to cell cycle arrest and DNA repair, apoptosis, or cellular senescence. More specifically, initial genomic insults lead to p53 stabilization and nuclear localization where transient cell cycle arrest can be quickly activated, allowing damaged DNA repair prior to replication. A signaling cascade can be activated by p53 in case of extreme and irreversible DNA damage to induce programmed cell death through transcription of different proapoptotic factors.

UV radiation induces phospholipids peroxidation in cellular membrane. Lipid peroxidation is a consequence of free primary radicals (ROS). This, in turn, leads to the generation of polar products and increase the membrane dielectric constant and capacitance. An important consequence of this phenomenon is the alteration of transport particles across the membrane (Strässle *et al*., 1991)

During the Fenton reaction, singlet oxygen directly initiates lipid peroxides and hydrogen peroxides indirectly initiate hydroxyl radicals (Halliwell & Gutteridge, 1999).

Cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence are induced by the p53 tumor suppressor pathway upon activation by genotoxic stress. This pathway works mostly through transactivation of different downstream targets, for example, p21 cell cycle inhibitor, required for short-term cell cycle arrest or long-term cellular senescence, or other proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) (Tavana *et al*., 2010). Yet, the mechanism that regulates the switching from cell cycle arrest to apoptosis is still unknown. In case of extreme or irreversible damage, p53 can additionally activate a signaling cascade to induce apoptosis through transcription of pro-apoptotic genes, most particularly p53-upregulated modulator of apoptosis (PUMA) and trans-repression of anti-apoptotic genes including Bcl-2. Programmed cell death directly protects cells against the accumulation of genomic instability that could lead to tumorigenesis.

Fig. 1. Schematic representation. Photobiological effects of ultraviolet radiation on human skin cells

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts 443

0.02% ethylene diamine tetra acetic acid (EDTA; Sigma Chemical Co., Saint Louis, MO,

Fig. 2. Optical microscopy. Primary human skin fibroblast culture. Hematoxilin and eosin

Cells were rinsed in PBS. The PBS was then removed and a thin layer of buffer was left on top of the coverslip. Fibroblasts were irradiated in culture dishes in a 10cm2 field using a UV

Primary human skin fibroblast culture were used after harvesting by trypsinization [0.025% trypsin, 0.02% ethylene diamine tetra acetic acid (EDTA; Sigma Chemical Co., Saint Louis, MO, USA) in PBS]. The cells were washed 3 times with phosphate-buffered saline (PBS). Human fibroblasts were plated on glass coverslips, xed in 2% paraformaldehyde for 10 minutes at 4°C, washed 3 times in PBS, and washed twice in PBS with 50 mmol/L NH4Cl. Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C and stained with a combination of fluorescent dyes. Filaments of cytoskeleton immunostained with phalloidin conjugated fluorescent with Alexa Fluor 594 (red) - Molecular Probe, were used to identify actin filaments F inside the cells. Phalloidin (1:500) incubation was performed in PBS containing 10% normal bovine serum and 0.1% saponin. Nuclei were counter stained with blue - fluorescent DNA stain DAPI (4\_6 diamidino-2-phenylindole) 1:10000 (catalog #D1036; Molecular Probes, Invitrogen, Carlsbad, CA ), and excited using a 750nm multiphoton source (two simultaneous photon excitations at 375nm). The images are a composite of three images acquired using filter sets appropriate for blue and red fluorescence, on a Zeiss confocal microscope (LSM 510,

chamber (with 6 UV F40 Philips lamps) in exposure times of 30 and 60 minutes.

USA) in PBS].

staining

Germany).

**3.2 Ultraviolet irradiation** 

**3.3 Immunofluorescence labeling** 

Senescence, an irreversible cell cycle arrest, can also be induced by DNA damage. p21, the cyclin-dependent kinase inhibitor, plays an important role in cell cycle checkpoint regulation and induction of cellular senescence, thus being one key p53 target. After DNA damage, p21 is commonly transactivated and induces G1arrest by inhibiting the cyclinE/CDK2 complex. (Campisi, 2009). Many different stimuli can induce cellular senescence including telomere shortening (replicative senescence), oncogenic signaling (oncogene-induced senescence), or stress/DNA damage irrespectively of the two previous signaling pathways (premature senescence) (Campisi, 2009). Despite the stimuli, cellular senescence and apoptosis are somewhat equivalent in preventing genomic instability and consequently inhibiting tumor formation (Van Nguyen, 2007). Upon UV exposure, p48 mRNA levels strongly depended on basal p53 expression and increased even more after DNA damage in a p53-dependent manner thus pointing as the link between p53 and the nucleotide excision repair apparatus (Hwang *et al*., 1999).

#### **2. Objective**

The objective of this study was to investigate modifications in cytoskeleton through the formation of blebs and apoptosis in cultured human fibroblasts by confocal microscopy and flow cytometry.

#### **3. Methods**

This study was performed in accordance with the ethical standards laid down in the updated version of the 1964 Declaration of Helsinki and was approved by the Research Ethics Committee of the Federal University of São Paulo. All patients signed a free and informed consent form. Samples of normal adult human skin (6 women, 18-50 years, skin phototype Fitzpatrick class. III-IV) were obtained as discarded tissue from trunk cosmetic surgery.

#### **3.1 Fibroblast culture**

Primary human skin fibroblast culture was done by explant. Fragments were placed in 15 ml conic tubes and exhaustively rinsed (six times) with 10 ml PBS (Phosphate-Buffered Saline, Cultilab, Campinas, SP, Brazil) containing penicillin (100 Ul/ml, Gibco, Carlsbad, CA, USA) and streptomycin (100μg/ml, Gibco) under vigorous agitation, changing tubes and PBS at each repetition. Then, fragments were transferred to 60 mm² diameter Petri dishes, in grid areas scratched with a scalpel. Dishes were left semi-opened in the laminar flow for 20 min, for the fragments to adhere to its surface. Then, 6 ml of DMEM (Dulbecco's Modified Eagle's Medium, Cultilab) supplemented with 10% FBS (Fetal Bovine Serum, Cultilab), 1% glutamine, penicillin (100 UI/ml, Gibco) and streptomycin (100 μg/ml, Gibco) were carefully added to each plate. Plates were kept in humidified incubator (37ºC, 95% O2, 5%CO2).

Culture medium was changed every two days and a few days after establishing the primary culture, spindle-like cells were seen proliferating from the edges of the explanted tissue, regarded as culturing fibroblasts. Fibroblast satisfactory proliferation was observed in approximately 7-14 days and subculturing (passage) was performed when cellular confluence reached approximately 80% at the Petri dish. For all experiments, cells from passages one to five (Figure 2) were used after harvesting by trypsinization [0.025% trypsin, 0.02% ethylene diamine tetra acetic acid (EDTA; Sigma Chemical Co., Saint Louis, MO, USA) in PBS].

Fig. 2. Optical microscopy. Primary human skin fibroblast culture. Hematoxilin and eosin staining

#### **3.2 Ultraviolet irradiation**

442 Flow Cytometry – Recent Perspectives

Senescence, an irreversible cell cycle arrest, can also be induced by DNA damage. p21, the cyclin-dependent kinase inhibitor, plays an important role in cell cycle checkpoint regulation and induction of cellular senescence, thus being one key p53 target. After DNA damage, p21 is commonly transactivated and induces G1arrest by inhibiting the cyclinE/CDK2 complex. (Campisi, 2009). Many different stimuli can induce cellular senescence including telomere shortening (replicative senescence), oncogenic signaling (oncogene-induced senescence), or stress/DNA damage irrespectively of the two previous signaling pathways (premature senescence) (Campisi, 2009). Despite the stimuli, cellular senescence and apoptosis are somewhat equivalent in preventing genomic instability and consequently inhibiting tumor formation (Van Nguyen, 2007). Upon UV exposure, p48 mRNA levels strongly depended on basal p53 expression and increased even more after DNA damage in a p53-dependent manner thus pointing as the link between p53 and the

The objective of this study was to investigate modifications in cytoskeleton through the formation of blebs and apoptosis in cultured human fibroblasts by confocal microscopy and

This study was performed in accordance with the ethical standards laid down in the updated version of the 1964 Declaration of Helsinki and was approved by the Research Ethics Committee of the Federal University of São Paulo. All patients signed a free and informed consent form. Samples of normal adult human skin (6 women, 18-50 years, skin phototype Fitzpatrick class. III-IV) were obtained as discarded tissue from trunk cosmetic surgery.

Primary human skin fibroblast culture was done by explant. Fragments were placed in 15 ml conic tubes and exhaustively rinsed (six times) with 10 ml PBS (Phosphate-Buffered Saline, Cultilab, Campinas, SP, Brazil) containing penicillin (100 Ul/ml, Gibco, Carlsbad, CA, USA) and streptomycin (100μg/ml, Gibco) under vigorous agitation, changing tubes and PBS at each repetition. Then, fragments were transferred to 60 mm² diameter Petri dishes, in grid areas scratched with a scalpel. Dishes were left semi-opened in the laminar flow for 20 min, for the fragments to adhere to its surface. Then, 6 ml of DMEM (Dulbecco's Modified Eagle's Medium, Cultilab) supplemented with 10% FBS (Fetal Bovine Serum, Cultilab), 1% glutamine, penicillin (100 UI/ml, Gibco) and streptomycin (100 μg/ml, Gibco) were carefully added to each plate. Plates were kept in humidified incubator (37ºC, 95% O2,

Culture medium was changed every two days and a few days after establishing the primary culture, spindle-like cells were seen proliferating from the edges of the explanted tissue, regarded as culturing fibroblasts. Fibroblast satisfactory proliferation was observed in approximately 7-14 days and subculturing (passage) was performed when cellular confluence reached approximately 80% at the Petri dish. For all experiments, cells from passages one to five (Figure 2) were used after harvesting by trypsinization [0.025% trypsin,

nucleotide excision repair apparatus (Hwang *et al*., 1999).

**2. Objective** 

flow cytometry.

**3. Methods** 

5%CO2).

**3.1 Fibroblast culture** 

Cells were rinsed in PBS. The PBS was then removed and a thin layer of buffer was left on top of the coverslip. Fibroblasts were irradiated in culture dishes in a 10cm2 field using a UV chamber (with 6 UV F40 Philips lamps) in exposure times of 30 and 60 minutes.

#### **3.3 Immunofluorescence labeling**

Primary human skin fibroblast culture were used after harvesting by trypsinization [0.025% trypsin, 0.02% ethylene diamine tetra acetic acid (EDTA; Sigma Chemical Co., Saint Louis, MO, USA) in PBS]. The cells were washed 3 times with phosphate-buffered saline (PBS). Human fibroblasts were plated on glass coverslips, xed in 2% paraformaldehyde for 10 minutes at 4°C, washed 3 times in PBS, and washed twice in PBS with 50 mmol/L NH4Cl. Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C and stained with a combination of fluorescent dyes. Filaments of cytoskeleton immunostained with phalloidin conjugated fluorescent with Alexa Fluor 594 (red) - Molecular Probe, were used to identify actin filaments F inside the cells. Phalloidin (1:500) incubation was performed in PBS containing 10% normal bovine serum and 0.1% saponin. Nuclei were counter stained with blue - fluorescent DNA stain DAPI (4\_6 diamidino-2-phenylindole) 1:10000 (catalog #D1036; Molecular Probes, Invitrogen, Carlsbad, CA ), and excited using a 750nm multiphoton source (two simultaneous photon excitations at 375nm). The images are a composite of three images acquired using filter sets appropriate for blue and red fluorescence, on a Zeiss confocal microscope (LSM 510, Germany).

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts 445

directions. Samples were examined by fluorescence-activated cell sorter (FACS) analysis, and the results were analyzed using Cell-Quest software (Becton Dickinson, San Jose, CA)

Briefly, normal human fibroblast cells from cultures with increasing passage number were collected and re-suspended in a buffer saline (PBS) containing 0.1% sodium azide (Sigma) containing 20 mM HEPES (pH 7.5), the cells were homogenized and centrifuged at 10,000 x g for 5 min. For analysis of caspase 3 and p53 expression, cells were xed in 2% paraformaldehyde for 10 minutes at 4°C, washed 3 times in PBS, then washed twice in PBS with 50 mmol/L NH4Cl. Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C. The rst primary antibody incubation (antip53 (SER 15) or anti–cleaved caspase 3) was performed in PBS containing 10% normal bovine serum and 0.1% saponin. Aliquots were then incubated for 60 minutes with anticaspase 3 and p53 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), final dilution 1:800, or rabbit IgG as a control, followed by washing in PBS containing 0.1% saponin 3 times for 5 minutes each at 22°C. Cells were then incubated with the rst uorochromeconjugated secondary antibodies Alexa 488 and 594 diluted 1:1600, and incubation was

The results obtained were analyzed using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls Multiple Range Test. Data were analyzed by

Skin cells exposure to solar radiation may result in biological consequences, one of the most important being skin DNA photodamage due to sunlight ultraviolet (UV) radiation. Wavelengths in the UVB range are absorbed by DNA and can induce mutagenesis. It has been suggested that p53-independent mechanisms of killing tumor cells may not involve programmed cell death and could be a result of induced mechanical damage, rather than

Ultraviolet A radiation (UVA, 320–400 nm), an oxidizing component of sunlight, exerts its biological effect mainly by producing reactive oxygen species (ROS) which cause biological damage in exposed tissues, including the lipid bilayer, via iron-catalyzed oxidative reactions (Halliwell & Gutteridge, 1999; Tyrrell, 1990). Membrane alterations induced by UV irradiation were determined, such as MDA concentration increase, which indicates lipid peroxidation levels (methods previously described -Figure 3). The UV radiation effects in the cellular production of ROS were indirectly determined by the ratio: (MDA concentration / total amount of fibroblasts) at the sample. Analyses of the lipid peroxidation by measuring the products that react with Tiobarbituric Acids (TBARs) normalizing the obtained MDA (malondialdehyde) results by the number of cells in the sample (Figure 4). A significant MDA increase was observed, of about 45.0 % after 30 min of UV exposure and 130% after 60

(Vermes *et al*., 1995).

**3.7 Statistical analysis** 

GraphPad Prism v.3.0 software.

**4. Results and discussion** 

apoptosis (Funkel, 1999).

min of UV exposure.

**3.6 Flow cytometric analysis of caspase 3 and p53** 

performed for 40 minutes at 37°C in the dark (Danova *et al*., 1990).

#### **3.4 Determination of MDA-TBA levels**

Taking the 1h time-point, which proved to be optimal for the determination of MDA increase, we then studied dose kinetics. Fibroblasts were exposed to a series of single doses UV irradiation in exposure times of 30 and 60 minutes. Markedly elevated MDA concentrations in the UV and TBARs–MDA complex concentrations were determined by high-performance liquid chromatography (HPLC) as described by Gueguen *et al*., 2002. The MDA-TBA test, which is the colorimetric reaction of malondialdehyde and thiobarbituric acid in acid solution, was used to determine the MDA levels. HPLC was used after the formation of the MDA-TBA complex (Figure 3) to assess the concentration of the complex based on a known standard curve. After heating at 95 ºC for 60 min, the MDA-TBA chromogen was fluorometrically analyzed using a reversed-phase C18 column HPLC and a wavelength of 532 nm. The MDA-TBA method was previously described by Chirico *et al*. (1993). MDA levels were expressed in relation to the total cellular lysate protein amount, which was assessed using Bradford's method (Bradford, 1976).

Fig. 3. Absorbance spectra for MDA – TBA chromogen complex standards in Thiobarbituric Acid Reactions (TBARs). Malondialdehyde (MDA) is a very effective method for determining lipid peroxidation levels in fibroblasts exposed to ultraviolet radiation. The standards absorption peaks of the inserted curve were highly linear in the range of 0 to 10nmoles/mL with maximum absorption at 532nm

#### **3.5 Apoptosis assay**

Flow cytometry technique, using propidium iodide, was used to detect apoptosis in fibroblast culture of human skin exposed to UV radiation (Nicoletti *et al*., 1991).

Human fibroblasts were labeled with annexin V-FITC (Roche), which bind to phosphatidylserine at the cell surface of apoptotic cells, and propidium iodide (PI; Sigma Aldrich), was used as a marker of cell membrane permeability according to manufacturer's directions. Samples were examined by fluorescence-activated cell sorter (FACS) analysis, and the results were analyzed using Cell-Quest software (Becton Dickinson, San Jose, CA) (Vermes *et al*., 1995).

#### **3.6 Flow cytometric analysis of caspase 3 and p53**

Briefly, normal human fibroblast cells from cultures with increasing passage number were collected and re-suspended in a buffer saline (PBS) containing 0.1% sodium azide (Sigma) containing 20 mM HEPES (pH 7.5), the cells were homogenized and centrifuged at 10,000 x g for 5 min. For analysis of caspase 3 and p53 expression, cells were xed in 2% paraformaldehyde for 10 minutes at 4°C, washed 3 times in PBS, then washed twice in PBS with 50 mmol/L NH4Cl. Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C. The rst primary antibody incubation (antip53 (SER 15) or anti–cleaved caspase 3) was performed in PBS containing 10% normal bovine serum and 0.1% saponin. Aliquots were then incubated for 60 minutes with anticaspase 3 and p53 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), final dilution 1:800, or rabbit IgG as a control, followed by washing in PBS containing 0.1% saponin 3 times for 5 minutes each at 22°C. Cells were then incubated with the rst uorochromeconjugated secondary antibodies Alexa 488 and 594 diluted 1:1600, and incubation was performed for 40 minutes at 37°C in the dark (Danova *et al*., 1990).

#### **3.7 Statistical analysis**

444 Flow Cytometry – Recent Perspectives

Taking the 1h time-point, which proved to be optimal for the determination of MDA increase, we then studied dose kinetics. Fibroblasts were exposed to a series of single doses UV irradiation in exposure times of 30 and 60 minutes. Markedly elevated MDA concentrations in the UV and TBARs–MDA complex concentrations were determined by high-performance liquid chromatography (HPLC) as described by Gueguen *et al*., 2002. The MDA-TBA test, which is the colorimetric reaction of malondialdehyde and thiobarbituric acid in acid solution, was used to determine the MDA levels. HPLC was used after the formation of the MDA-TBA complex (Figure 3) to assess the concentration of the complex based on a known standard curve. After heating at 95 ºC for 60 min, the MDA-TBA chromogen was fluorometrically analyzed using a reversed-phase C18 column HPLC and a wavelength of 532 nm. The MDA-TBA method was previously described by Chirico *et al*. (1993). MDA levels were expressed in relation to the total cellular lysate protein amount,

Fig. 3. Absorbance spectra for MDA – TBA chromogen complex standards in Thiobarbituric Acid Reactions (TBARs). Malondialdehyde (MDA) is a very effective method for determining lipid peroxidation levels in fibroblasts exposed to ultraviolet radiation. The standards absorption peaks of the inserted curve were highly linear in the range of 0 to 10nmoles/mL

Flow cytometry technique, using propidium iodide, was used to detect apoptosis in

Human fibroblasts were labeled with annexin V-FITC (Roche), which bind to phosphatidylserine at the cell surface of apoptotic cells, and propidium iodide (PI; Sigma Aldrich), was used as a marker of cell membrane permeability according to manufacturer's

fibroblast culture of human skin exposed to UV radiation (Nicoletti *et al*., 1991).

**3.4 Determination of MDA-TBA levels** 

with maximum absorption at 532nm

**3.5 Apoptosis assay** 

which was assessed using Bradford's method (Bradford, 1976).

The results obtained were analyzed using a one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls Multiple Range Test. Data were analyzed by GraphPad Prism v.3.0 software.

#### **4. Results and discussion**

Skin cells exposure to solar radiation may result in biological consequences, one of the most important being skin DNA photodamage due to sunlight ultraviolet (UV) radiation. Wavelengths in the UVB range are absorbed by DNA and can induce mutagenesis. It has been suggested that p53-independent mechanisms of killing tumor cells may not involve programmed cell death and could be a result of induced mechanical damage, rather than apoptosis (Funkel, 1999).

Ultraviolet A radiation (UVA, 320–400 nm), an oxidizing component of sunlight, exerts its biological effect mainly by producing reactive oxygen species (ROS) which cause biological damage in exposed tissues, including the lipid bilayer, via iron-catalyzed oxidative reactions (Halliwell & Gutteridge, 1999; Tyrrell, 1990). Membrane alterations induced by UV irradiation were determined, such as MDA concentration increase, which indicates lipid peroxidation levels (methods previously described -Figure 3). The UV radiation effects in the cellular production of ROS were indirectly determined by the ratio: (MDA concentration / total amount of fibroblasts) at the sample. Analyses of the lipid peroxidation by measuring the products that react with Tiobarbituric Acids (TBARs) normalizing the obtained MDA (malondialdehyde) results by the number of cells in the sample (Figure 4). A significant MDA increase was observed, of about 45.0 % after 30 min of UV exposure and 130% after 60 min of UV exposure.

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts 447

Fig. 5. Confocal microscopy. Cultured human skin fibroblasts. Control group. Cellular localization of actin filaments and nuclei. A) Actin filaments immunostained with phalloidin conjugated with Alexa Fluor 594 (red). B) Cell nuclei stained with DAPI (blue), showing characteristics of nuclear and cytoskeleton integrity. High cellularity was also observed.

Fig. 6. Confocal microscopy. Cultured human skin fibroblasts. Cellular localization of actin filaments and nuclei. Cells exposed to UV radiation for 30 min (1A, 1B, 1C) or 60 min (2A, 2B, 2C). 1A) Actin filaments immunostained with phalloidin conjugated with Alexa Fluor 594 (red). The occurrence of blebbing can be observed. 1B) Cell nuclei stained with DAPI (blue), showing characteristics of nuclear and cytoskeleton integrity. 1C) Overlapped images A and B. 2A) Actin filaments immunostained with phalloidin conjugated with Alexa Fluor 594 (red). 2B) Pyknotic nuclei (\*) and nuclear fragmentation (arrow) were observed. 2C)

C) Overlapped images A and B

Overlapped images A and B

Fig. 4. Effect of UV radiation in the cellular production of oxygen reactive species measured by ratio: MDA concentration by total number of fibroblast in the sample. Histograms values differ significantly from each other. \*Data analyzed with one-way ANOVA followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least four different experiments

Similar results are also described by several studies demonstrating that low UVA radiation doses can induce lipid peroxidation in membranes of both human broblasts and keratinocytes via pathways involving singlet oxygen and iron (Morliere *et al*, 1991).

Looking from a different angle, cells also have repair mechanisms to respond to DNA damage, and at least two different mechanisms are responsible for UVA-induced DNA damage repair. The primary process that removes bulky damage is the nucleotide excision repair pathway. Small lesions induced by ROS are mostly processed by base excision repair pathway. On the other hand, highly damaged cells may undergo cell cycle arrest, apoptosis and senescence (Hazane *et al*., 2006). Our results are consistent with those of Shindo *et al.* (1994) who investigated antioxidant molecules in crude extracts of human epidermis and dermis. In addition, Moysan *et al.* (1995), using cells from the same biopsy, found no link between UVA cytotoxicity and antioxidant capacity since SOD, catalase and GSH were identical in both cells and GSH-Px was higher in fibroblasts (Degterev *et al*., 2008). Other authors, however, have found more antioxidant molecules in fibroblasts than in keratinocytes. Yohn *et al.* (1991), using cells from different donors, found increased GSH-Px, SOD and catalase in fibroblasts compared to keratinocytes, and in keratinocytes compared to melanocytes (Huang *et al*., 2008).

Several *in vitro* and *in vivo* studies on skin cells have demonstrated that UV radiation can damage many molecules and structures (Matsumura & Ananthaswamy, 2004). Corroborating these results, morphological analysis by confocal fluorescence microscopy of fibroblasts group control showed characteristics of nuclear and cytoskeleton integrity. High cellularity was also observed (Figure 5). In contrast, exposed to UV for 30 and 60 minutes showed changes in the actin filaments arrangement of the cellular cytoskeleton. Groups irradiated for 30 and 60 min presented disruption of the actin filaments, with the formation of blebbing and nuclear fragmentation as a consequence of the ultraviolet radiation (Figure 6).

Fig. 4. Effect of UV radiation in the cellular production of oxygen reactive species measured by ratio: MDA concentration by total number of fibroblast in the sample. Histograms values differ significantly from each other. \*Data analyzed with one-way ANOVA followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least

Similar results are also described by several studies demonstrating that low UVA radiation doses can induce lipid peroxidation in membranes of both human broblasts and

Looking from a different angle, cells also have repair mechanisms to respond to DNA damage, and at least two different mechanisms are responsible for UVA-induced DNA damage repair. The primary process that removes bulky damage is the nucleotide excision repair pathway. Small lesions induced by ROS are mostly processed by base excision repair pathway. On the other hand, highly damaged cells may undergo cell cycle arrest, apoptosis and senescence (Hazane *et al*., 2006). Our results are consistent with those of Shindo *et al.* (1994) who investigated antioxidant molecules in crude extracts of human epidermis and dermis. In addition, Moysan *et al.* (1995), using cells from the same biopsy, found no link between UVA cytotoxicity and antioxidant capacity since SOD, catalase and GSH were identical in both cells and GSH-Px was higher in fibroblasts (Degterev *et al*., 2008). Other authors, however, have found more antioxidant molecules in fibroblasts than in keratinocytes. Yohn *et al.* (1991), using cells from different donors, found increased GSH-Px, SOD and catalase in fibroblasts compared to keratinocytes, and in keratinocytes compared

Several *in vitro* and *in vivo* studies on skin cells have demonstrated that UV radiation can damage many molecules and structures (Matsumura & Ananthaswamy, 2004). Corroborating these results, morphological analysis by confocal fluorescence microscopy of fibroblasts group control showed characteristics of nuclear and cytoskeleton integrity. High cellularity was also observed (Figure 5). In contrast, exposed to UV for 30 and 60 minutes showed changes in the actin filaments arrangement of the cellular cytoskeleton. Groups irradiated for 30 and 60 min presented disruption of the actin filaments, with the formation of blebbing

and nuclear fragmentation as a consequence of the ultraviolet radiation (Figure 6).

keratinocytes via pathways involving singlet oxygen and iron (Morliere *et al*, 1991).

four different experiments

to melanocytes (Huang *et al*., 2008).

Fig. 5. Confocal microscopy. Cultured human skin fibroblasts. Control group. Cellular localization of actin filaments and nuclei. A) Actin filaments immunostained with phalloidin conjugated with Alexa Fluor 594 (red). B) Cell nuclei stained with DAPI (blue), showing characteristics of nuclear and cytoskeleton integrity. High cellularity was also observed. C) Overlapped images A and B

Fig. 6. Confocal microscopy. Cultured human skin fibroblasts. Cellular localization of actin filaments and nuclei. Cells exposed to UV radiation for 30 min (1A, 1B, 1C) or 60 min (2A, 2B, 2C). 1A) Actin filaments immunostained with phalloidin conjugated with Alexa Fluor 594 (red). The occurrence of blebbing can be observed. 1B) Cell nuclei stained with DAPI (blue), showing characteristics of nuclear and cytoskeleton integrity. 1C) Overlapped images A and B. 2A) Actin filaments immunostained with phalloidin conjugated with Alexa Fluor 594 (red). 2B) Pyknotic nuclei (\*) and nuclear fragmentation (arrow) were observed. 2C) Overlapped images A and B

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts 449

Fig. 8. Mean percentage ± of apoptotic cells in groups control and 30min and 60min after exposure to UV radiation. Data are the means of triplicate assays of one experiment representative of three that gave similar results. A) Total number of viable cells and B) Percentage of apoptotic cells. \*Data analyzed with one-way **ANOVA** followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least four different

DNA is a critical target because its alteration can ultimately lead to the development of skin cancer (Matsumura *et al*., 2004). In addition, Skin DNA photodamage activates the signaling pathway of cell death by apoptosis. Apoptosis is a crucial mechanism in eliminating cells with unrepaired DNA damage and preventing carcinogenesis (or preventing the formation

Apoptosis is characterized by a p53-dependent induction of pro-apoptotic proteins, leading to permeabilization of the outer mitochondrial membrane, release of apoptogenic factors into the cytoplasm, activation of caspases (cysteine-aspartic proteases) and subsequent cleavage of various cellular proteins. Apoptogenic effects include chromatin condensation

p53 levels increased about 40% after 30 min of UV exposure and about 60% after 60 min of

Previous studies indicated that BimL was involved in UV-induced apoptosis, but it remains unclear whether Bim directly activates Bax or if this activation occurs via the release of pro-survival factors (antiapoptotic) such as Bcl-xL. In recent studies, Wang *et al*. (2009) determined the interactions between BimL and Bax/Bcl-xL during UV-induced apoptosis. Caspases have a major role in apoptosis. They are synthesized as inactive proenzymes that become activated by cleavage. Procaspase 3 is a constitutive proenzyme activated by cleavage during apoptosis. (Cohen, 1997). Caspase-3 is the most important protease in the caspase-dependent apoptosis pathway, as it is required for chromatin condensation and fragmentation (Porter & Jänicke, 1999). Poly-ADP ribose polymerase (PARP-1) is a major target of caspase-3, since cleavage-mediated inactivation of PARP-1 preserves cellular ATP

Regarding the caspases, the resulting enzyme is able to cleave several aspartate residues of many target proteins, after a DEVD sequence common to all caspases 3 and 7 substrates

and exposure of phosphatidylserine on the cell membrane surface (Meier *et al*., 2007).

experiments

of malignant tumors).

UV exposure (Figure 9).

that is required for apoptosis (Bouchard *et al*., 2003).

In addition, skin fibroblasts viability, stained by propidium iodide (PI), was analyzed by flow cytometry. Viable cells were characterized by a structurally intact cell membrane and no PI uptake. In contrast, dead cells (necrosis or late apoptotic cells) were characterized by loss of the integrity of their membranes and were stained by PI. At all UV radiation tested doses, the amount of viable cells was reduced, as verified by PI staining. The amount of viable fibroblasts was dramatically reduced by UV radiation at all tested doses/exposure times, about 80% after 30 min of exposure and 30% after 60 min of exposure (Figures 7A and 8A).

There are strong evidences that skin cancer can be developed as a result of ultraviolet radiation, which is directly associated to the TP53-gene tumor mutation.

To further investigate whether p53 is involved in the apoptosis induced by UV, cells were first stained for membrane-exposed phosphatidylserine using annexin-V conjugated to fluorescein (FITC). There was a significant increase of the number of apoptotic cells: about 21.0 % (30 min) and 50% (60 min) after irradiation (Figures 7B and 8B) and (Figures 7C and 8C), respectively.

Fig. 7. Contour diagram of PI flow cytometry of cultured fibroblasts for groups: A) Control; B) UV irradiated for 30 min and C) UV irradiated for 60 min. The lower left quadrant of the cytograms shows the viable cells, which excluded PI. The upper right quadrants represent the apoptotic cells showing PI uptake. Panel (B) shows cells number (%) for apoptosis and necrosis 30 minutes after exposure to ultraviolet radiation. Panel (C) shows cells number (%) for apoptosis and necrosis 60 minutes after exposure to ultraviolet radiation. Data are representative of 04 independent experiments

Ultraviolet radiation is a carcinogenic agent for the skin. Even though being a tumor suppressor gene, details are still needed in order to understand the signaling mechanisms of skin cell death induced by UV radiations, which can lead to cancer and/or cell aging.

DNA alteration can ultimately lead to the development of skin cancer, so DNA itself is a critical target (Matsumura *et al*., 2004). Skin DNA photodamage activates the signaling pathway of cell death by apoptosis. Apoptosis is a crucial mechanism in eliminating cells with unrepaired DNA damage and preventing carcinogenesis.

In addition, skin fibroblasts viability, stained by propidium iodide (PI), was analyzed by flow cytometry. Viable cells were characterized by a structurally intact cell membrane and no PI uptake. In contrast, dead cells (necrosis or late apoptotic cells) were characterized by loss of the integrity of their membranes and were stained by PI. At all UV radiation tested doses, the amount of viable cells was reduced, as verified by PI staining. The amount of viable fibroblasts was dramatically reduced by UV radiation at all tested doses/exposure times, about 80% after 30 min of exposure and 30% after 60 min of exposure (Figures 7A and 8A). There are strong evidences that skin cancer can be developed as a result of ultraviolet

To further investigate whether p53 is involved in the apoptosis induced by UV, cells were first stained for membrane-exposed phosphatidylserine using annexin-V conjugated to fluorescein (FITC). There was a significant increase of the number of apoptotic cells: about 21.0 % (30 min) and 50% (60 min) after irradiation (Figures 7B and 8B) and (Figures 7C and

Fig. 7. Contour diagram of PI flow cytometry of cultured fibroblasts for groups: A) Control; B) UV irradiated for 30 min and C) UV irradiated for 60 min. The lower left quadrant of the cytograms shows the viable cells, which excluded PI. The upper right quadrants represent the apoptotic cells showing PI uptake. Panel (B) shows cells number (%) for apoptosis and necrosis 30 minutes after exposure to ultraviolet radiation. Panel (C) shows cells number (%) for apoptosis and necrosis 60 minutes after exposure to ultraviolet radiation. Data are

Ultraviolet radiation is a carcinogenic agent for the skin. Even though being a tumor suppressor gene, details are still needed in order to understand the signaling mechanisms of skin cell death induced by UV radiations, which can lead to cancer and/or cell aging.

DNA alteration can ultimately lead to the development of skin cancer, so DNA itself is a critical target (Matsumura *et al*., 2004). Skin DNA photodamage activates the signaling pathway of cell death by apoptosis. Apoptosis is a crucial mechanism in eliminating cells

representative of 04 independent experiments

with unrepaired DNA damage and preventing carcinogenesis.

radiation, which is directly associated to the TP53-gene tumor mutation.

8C), respectively.

Fig. 8. Mean percentage ± of apoptotic cells in groups control and 30min and 60min after exposure to UV radiation. Data are the means of triplicate assays of one experiment representative of three that gave similar results. A) Total number of viable cells and B) Percentage of apoptotic cells. \*Data analyzed with one-way **ANOVA** followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least four different experiments

DNA is a critical target because its alteration can ultimately lead to the development of skin cancer (Matsumura *et al*., 2004). In addition, Skin DNA photodamage activates the signaling pathway of cell death by apoptosis. Apoptosis is a crucial mechanism in eliminating cells with unrepaired DNA damage and preventing carcinogenesis (or preventing the formation of malignant tumors).

Apoptosis is characterized by a p53-dependent induction of pro-apoptotic proteins, leading to permeabilization of the outer mitochondrial membrane, release of apoptogenic factors into the cytoplasm, activation of caspases (cysteine-aspartic proteases) and subsequent cleavage of various cellular proteins. Apoptogenic effects include chromatin condensation and exposure of phosphatidylserine on the cell membrane surface (Meier *et al*., 2007).

p53 levels increased about 40% after 30 min of UV exposure and about 60% after 60 min of UV exposure (Figure 9).

Previous studies indicated that BimL was involved in UV-induced apoptosis, but it remains unclear whether Bim directly activates Bax or if this activation occurs via the release of pro-survival factors (antiapoptotic) such as Bcl-xL. In recent studies, Wang *et al*. (2009) determined the interactions between BimL and Bax/Bcl-xL during UV-induced apoptosis.

Caspases have a major role in apoptosis. They are synthesized as inactive proenzymes that become activated by cleavage. Procaspase 3 is a constitutive proenzyme activated by cleavage during apoptosis. (Cohen, 1997). Caspase-3 is the most important protease in the caspase-dependent apoptosis pathway, as it is required for chromatin condensation and fragmentation (Porter & Jänicke, 1999). Poly-ADP ribose polymerase (PARP-1) is a major target of caspase-3, since cleavage-mediated inactivation of PARP-1 preserves cellular ATP that is required for apoptosis (Bouchard *et al*., 2003).

Regarding the caspases, the resulting enzyme is able to cleave several aspartate residues of many target proteins, after a DEVD sequence common to all caspases 3 and 7 substrates

Biological Effects Induced by Ultraviolet Radiation in Human Fibroblasts 451

(a) (b)

(c) (d)

four different experiments

Fig. 10. Activation of caspase 3 by UV can be followed by ow cytometry (FCM) of cultured fibroblasts for groups: A) control (upper left set of panel – figure 10A - Black line) and UV irradiated for 30 min (right set of figure 10B – Blue line) and UV irradiated for 60 min (right set of 10C – orange line). The fibroblasts treated in 2% paraformaldehyde are the same as those shown in figure 05 (control group) and figure 06 (UV irradiated groups). Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C and stained with anti–cleaved caspase 3 antibodies at figures (10A), (10B), and (10C) after the beginning of the experiment and analyzed by FCM. A control performed with an irrelevant antibody is shown 10A. The percentage of cells exhibiting active caspase 3 conjugated with FITC is indicated on each histogram. The results from one representative experiment of four experiments performed are shown. The numbers indicate the percentages of positive cells and uorescence intensity. Histogram overlays show the FL1 (green uorescence) intensity corresponding to a given caspase 3:(blue line – UV irradiated for 30min and red line – UV irradiated for 60min compared to the intensity for the control (black line). 10D Mean percentage ± of cells exhibiting active caspase 3 in groups control (figure 9A – green line) and groups UV irradiated for 30 min (figure 9B – Black line) and UV irradiated for 60 min (figure 9C – red line). Data are the means of triplicate assays of one experiment representative of three that gave similar results. A) Total number cells fibroblasts exhibiting active caspase 3 antibodies at figures (10A), (10B), and (10C). Histograms values differ significantly from each other. \*Data analyzed with one-way ANOVA followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least

Fig. 9. Flow cytometry (FCM) analysis of p53 protein accumulation control (upper left set of panel – figure 9A – green line) and / or activation by UV can be followed of cultured fibroblasts for groups: UV irradiated for 30 min (right set of figure 9B – Black line) and UV irradiated for 60 min (right set of figure 9C – red line). The fibroblasts treated in 2% paraformaldehyde are the same as those shown in Figure 05 (control group) and figure 06 (UV irradiated groups). Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C and stained with anti-p53 (SER 15) antibodies at figures (9A), (9B), and (9C) after the beginning of the experiment and analyzed by FCM. A control performed with an irrelevant antibody is shown figure 9A. The percentage of cells exhibiting active p53 conjugated with FITC is indicated on each histogram. The results from one representative experiment of four experiments performed are shown. The numbers indicate the percentages of positive cells and uorescence intensity. Histogram overlays show the FL1 (green uorescence) intensity corresponding to a given p53 (black line – UV irradiated for 30min and red line – UV irradiated for 60min) compared to the intensity for the control (green line). 9D Mean percentage ± of cells exhibiting active p53 in groups control (figure 9A – green line) and groups UV irradiate for 30 min (figure 9B – Black line) and UV irradiated for 60 min (figure 9C – red line). Data are the means of triplicate assays of one experiment representative of three that gave similar results. A) Total number cells fibroblasts exhibiting active p53 antibodies at figures (9A), (9B), and (9C). Histograms values differ significantly from each other. \*Data analyzed with one-way ANOVA followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least four different experiments

(a) (b)

(c) (d)

four different experiments

Fig. 9. Flow cytometry (FCM) analysis of p53 protein accumulation control (upper left set of panel – figure 9A – green line) and / or activation by UV can be followed of cultured fibroblasts for groups: UV irradiated for 30 min (right set of figure 9B – Black line) and UV irradiated for 60 min (right set of figure 9C – red line). The fibroblasts treated in 2% paraformaldehyde are the same as those shown in Figure 05 (control group) and figure 06 (UV irradiated groups). Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C and stained with anti-p53 (SER 15) antibodies at figures (9A), (9B), and (9C) after the beginning of the experiment and analyzed by FCM. A control performed with an irrelevant antibody is shown figure 9A. The percentage of cells exhibiting active p53 conjugated with FITC is indicated on each histogram. The results from one representative experiment of four experiments performed are shown. The numbers indicate the percentages of positive cells and uorescence intensity. Histogram overlays show the FL1 (green uorescence) intensity corresponding to a given p53 (black line – UV irradiated for 30min and red line – UV irradiated for 60min) compared to the intensity for the control (green line). 9D Mean percentage ± of cells exhibiting active p53 in groups control (figure 9A – green line) and groups UV irradiate for 30 min (figure 9B – Black line) and UV irradiated for 60 min (figure 9C – red line). Data are the means of triplicate assays of one experiment representative of three that gave similar results. A) Total number cells fibroblasts exhibiting active p53 antibodies at figures (9A), (9B), and (9C). Histograms values differ significantly from each other. \*Data analyzed with one-way ANOVA followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least

Fig. 10. Activation of caspase 3 by UV can be followed by ow cytometry (FCM) of cultured fibroblasts for groups: A) control (upper left set of panel – figure 10A - Black line) and UV irradiated for 30 min (right set of figure 10B – Blue line) and UV irradiated for 60 min (right set of 10C – orange line). The fibroblasts treated in 2% paraformaldehyde are the same as those shown in figure 05 (control group) and figure 06 (UV irradiated groups). Cells were permeabilized with 0.1% saponin in PBS containing 10% normal bovine serum for 30 minutes at 22°C and stained with anti–cleaved caspase 3 antibodies at figures (10A), (10B), and (10C) after the beginning of the experiment and analyzed by FCM. A control performed with an irrelevant antibody is shown 10A. The percentage of cells exhibiting active caspase 3 conjugated with FITC is indicated on each histogram. The results from one representative experiment of four experiments performed are shown. The numbers indicate the percentages of positive cells and uorescence intensity. Histogram overlays show the FL1 (green uorescence) intensity corresponding to a given caspase 3:(blue line – UV irradiated for 30min and red line – UV irradiated for 60min compared to the intensity for the control (black line). 10D Mean percentage ± of cells exhibiting active caspase 3 in groups control (figure 9A – green line) and groups UV irradiated for 30 min (figure 9B – Black line) and UV irradiated for 60 min (figure 9C – red line). Data are the means of triplicate assays of one experiment representative of three that gave similar results. A) Total number cells fibroblasts exhibiting active caspase 3 antibodies at figures (10A), (10B), and (10C). Histograms values differ significantly from each other. \*Data analyzed with one-way ANOVA followed by Newman Keuls (significance level p < 0.05). Values represent the mean ± SEM of at least four different experiments

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(DEVDase). Thus, active caspase 3 is a common effector protein in several apoptotic pathways, and it may be a good marker to detect (pre-) apoptotic cells by ow cytometry (Porter & Jänicke, 1999). Taking this into consideration, apoptosis was confirmed by determining the increased expression of cleaved caspase 3 after fibroblasts exposure to UV radiation. In this work we could verify a significant increase of cleaved caspase 3 levels, about 25.0 % after 30 min of UV exposure and 75% after 60 min of UV exposure (Figure 10).

Although caspases represent a significant component of the apoptotic pathway, there is indication that a caspase-independent apoptosis pathway also exists (Broker *et al*., 2005). This pathway involves the Apoptosis-Inducing Factor (AIF), which translocates from the mitochondria to the nucleus to cause chromatin condensation (Daugas *et al*., 2000).

Then again, genotoxic effects of solar UVA are mediated essentially by the activation of endogenous photosensitizers which generate a local oxidative stress. Depending on the dose and duration of exposure, UV-induced effects may occur, and DNA damage can lead to mutations and genetic instability. This is one of the reasons why sunlight overexposure increases the risk of skin cancer and DNA photolesions can also be involved in other skinspecic responses to UV radiation: erythema, immunosuppression, and melanogenesis (Matsumura & Ananthaswamy, 2004).

#### **5. Conclusion**

Damages occurring on DNA molecules not always induce mutagenesis. We should take in consideration many strong scientific evidences showing that specific activation molecular signaling pathways promote several different answers. Both the prolonged exposure time and the increase in the UV radiation dose were able to induce lipid peroxidation and cell death by apoptosis. Our results suggest that the major part of UV induced apoptosis cell death is caspase-dependent, although a minority of cells may die by a caspase-independent pathway, presumably apoptotic. In this work we also showed that p53 levels increased after UV exposure. In these circumstances, the action of UV radiation on skin cells still involves many issues depending on the cell type and on different cellular response pathways induced by phototoxic stress. Skin fibroblasts are surely sensitive to UV radiation, thus, from a better understanding of the molecular mechanisms triggered by the action of UV radiation on skin cells, it will be possible to work on improving skin radioprotection and attenuating the effects of sunlight exposure.

#### **6. Acknowledgment**

We would like to thank UNIFESP and UESC (collaborators) for their help in experiments with fibroblast cell culture. The authors gratefully acknowledge the financial support from FAPESP, FAPESB and CNPq grants.

#### **7. References**

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(DEVDase). Thus, active caspase 3 is a common effector protein in several apoptotic pathways, and it may be a good marker to detect (pre-) apoptotic cells by ow cytometry (Porter & Jänicke, 1999). Taking this into consideration, apoptosis was confirmed by determining the increased expression of cleaved caspase 3 after fibroblasts exposure to UV radiation. In this work we could verify a significant increase of cleaved caspase 3 levels, about 25.0 % after 30 min of UV exposure and 75% after 60 min of UV exposure (Figure 10). Although caspases represent a significant component of the apoptotic pathway, there is indication that a caspase-independent apoptosis pathway also exists (Broker *et al*., 2005). This pathway involves the Apoptosis-Inducing Factor (AIF), which translocates from the

mitochondria to the nucleus to cause chromatin condensation (Daugas *et al*., 2000).

(Matsumura & Ananthaswamy, 2004).

attenuating the effects of sunlight exposure.

FAPESP, FAPESB and CNPq grants.

**6. Acknowledgment** 

**7. References** 

**5. Conclusion** 

Then again, genotoxic effects of solar UVA are mediated essentially by the activation of endogenous photosensitizers which generate a local oxidative stress. Depending on the dose and duration of exposure, UV-induced effects may occur, and DNA damage can lead to mutations and genetic instability. This is one of the reasons why sunlight overexposure increases the risk of skin cancer and DNA photolesions can also be involved in other skinspecic responses to UV radiation: erythema, immunosuppression, and melanogenesis

Damages occurring on DNA molecules not always induce mutagenesis. We should take in consideration many strong scientific evidences showing that specific activation molecular signaling pathways promote several different answers. Both the prolonged exposure time and the increase in the UV radiation dose were able to induce lipid peroxidation and cell death by apoptosis. Our results suggest that the major part of UV induced apoptosis cell death is caspase-dependent, although a minority of cells may die by a caspase-independent pathway, presumably apoptotic. In this work we also showed that p53 levels increased after UV exposure. In these circumstances, the action of UV radiation on skin cells still involves many issues depending on the cell type and on different cellular response pathways induced by phototoxic stress. Skin fibroblasts are surely sensitive to UV radiation, thus, from a better understanding of the molecular mechanisms triggered by the action of UV radiation on skin cells, it will be possible to work on improving skin radioprotection and

We would like to thank UNIFESP and UESC (collaborators) for their help in experiments with fibroblast cell culture. The authors gratefully acknowledge the financial support from

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**23** 

*Portugal* 

**Immunophenotypic Characterization** 

Despite of being described more than one decade ago (Pittenger et al., 1999), the immunophenotypic profile of bone marrow mesenchymal stem cells (MSC) still not well documented. The difficulty in achieving a detailed phenotypic characterization is common in less-represented cell populations and/or populations lacking a specific known cell

The recent advances in flow cytometry technology and the emergence of new high-speed flow cytometers have given a valuable contribute to diminish this problem in two different (but complementary) aspects: 1) by reducing dramatically the acquisition time period, making it more reasonable to study minor cell populations; and 2) by increasing the number of parameters that can be analyzed per cell at the same time, which is critical to improve the immunophenotypic characterization of those not-well characterized cell populations that

A good example of the practical usefulness of such technical developments is the description of different cell compartments in the bone marrow CD34+ hematopoietic stem cell (HSC) population. Detailed studies on this minor bone marrow cell population demonstrated that each compartment is committed to a different hematopoietic cell lineage. An extensive immunophenotypic characterization of those CD34+ compartments allowed the development of protocols to easily and quickly identify, quantify and evaluate phenotypic aberrations and maturational blocks in those cells, which is decisive to the diagnosis, prognosis, or follow-up of a variety of hematological clonal diseases (del Cañizo

After the identification of a plastic-adherent bone marrow stromal cell population in 1976 by Friedenstein and colleagues and the first evidence of their multilineage potential (Pittenger et al., 1999) with subsequent confirmation of their stem cell nature, an increasing interest on these bone marrow MSC has emerged, mainly because of their promising therapeutic

et al., 2003; Lochem et al., 2004; Matarraz et al., 2008; Orfao et al. 2004).

**2. Bone marrow mesenchymal stem cells** 

**1. Introduction** 

marker, like bone marrow MSC.

lack a specific known marker.

applications.

**of Normal Bone Marrow Stem Cells** 

Paula Laranjeira, Andreia Ribeiro, Sandrine Mendes,

Ana Henriques, M. Luísa Pais and Artur Paiva

*Histocompatibility Center of Coimbra* 

hypersensitivity, enhanced immunosuppression and cellular senescence. *Cell Cycle*, Vol.9, pp.3328-3336, ISSN 1551-4005


### **Immunophenotypic Characterization of Normal Bone Marrow Stem Cells**

Paula Laranjeira, Andreia Ribeiro, Sandrine Mendes, Ana Henriques, M. Luísa Pais and Artur Paiva *Histocompatibility Center of Coimbra Portugal* 

#### **1. Introduction**

456 Flow Cytometry – Recent Perspectives

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Despite of being described more than one decade ago (Pittenger et al., 1999), the immunophenotypic profile of bone marrow mesenchymal stem cells (MSC) still not well documented. The difficulty in achieving a detailed phenotypic characterization is common in less-represented cell populations and/or populations lacking a specific known cell marker, like bone marrow MSC.

The recent advances in flow cytometry technology and the emergence of new high-speed flow cytometers have given a valuable contribute to diminish this problem in two different (but complementary) aspects: 1) by reducing dramatically the acquisition time period, making it more reasonable to study minor cell populations; and 2) by increasing the number of parameters that can be analyzed per cell at the same time, which is critical to improve the immunophenotypic characterization of those not-well characterized cell populations that lack a specific known marker.

A good example of the practical usefulness of such technical developments is the description of different cell compartments in the bone marrow CD34+ hematopoietic stem cell (HSC) population. Detailed studies on this minor bone marrow cell population demonstrated that each compartment is committed to a different hematopoietic cell lineage. An extensive immunophenotypic characterization of those CD34+ compartments allowed the development of protocols to easily and quickly identify, quantify and evaluate phenotypic aberrations and maturational blocks in those cells, which is decisive to the diagnosis, prognosis, or follow-up of a variety of hematological clonal diseases (del Cañizo et al., 2003; Lochem et al., 2004; Matarraz et al., 2008; Orfao et al. 2004).

#### **2. Bone marrow mesenchymal stem cells**

After the identification of a plastic-adherent bone marrow stromal cell population in 1976 by Friedenstein and colleagues and the first evidence of their multilineage potential (Pittenger et al., 1999) with subsequent confirmation of their stem cell nature, an increasing interest on these bone marrow MSC has emerged, mainly because of their promising therapeutic applications.

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 459

fluorochromes. Although the compensation is automatic, it is always revised by experienced

In order to detect cellular autofluorescence, a negative control was made for each sample, where the bone marrow sample was only stained for CD45 PO and CD34 PerCPcy5.5.

SSC and FSC light dispersion properties allow a good discrimination between viable and dead cells and the doublets were excluded based on FSC-Area *versus* FSC-Height

The immunophenotypic characterization of bone marrow MSC were performed in fresh EDTA-collected bone marrow samples from healthy individuals. After collection the

Whole bone marrow samples were stained for surface cell markers using a stain-lyse-andthen-wash direct immunofluorescence technique. 200 μl of whole bone marrow were aliquoted in different tubes and stained with the following combinations of monoclonal

FITC PE PerCPcy5.5 PEcy7 APC APCH7 PB PO

CD13 CD90

CD90 (5E10) BD Pharmingen

> CD133 (293C3) Miltenyi Biotec

HLA-A, B, C (G46-2.6) BD Pharmingen

HLA-DR (L243) BDB

CD29 (TS2/16) BioLegend

CD11b (ICRF44) BD Pharmingen



CD11b CD45

CD45 (HI30) Invitrogen

CD13 (Immu103.44) Beckman Coulter

CD13

<sup>5</sup>- CD73 CD24 CD13 CD90 - CD11b CD45 Table 1. Panel of monoclonal antibodies used for the bone marrow MSC characterization. FITC - fluorescein isothiocyanate; PE – phycoerythrin; PerCPcy5.5 - peridinin chlorophyll protein cyanine 5.5; PEcy7 - R-phycoerythrin cyanine 7; APC – allophycocyanin; APCH7 -

Data acquisition was performed in a FACSCanto II flow cytometer (BDB), using FACSDiva acquisition software (BDB). The total bone marrow cellularity of the whole sample was acquired (5 x 106 events, minimum) for each tube. Bone marrow MSC were identified as

Data analysis was performed using Infinicyt software (Cytognos, Salamanca, Spain).


staff at the end of the process.

**2.2.2 Material and methods** 

samples were stored at 4 ºC and processed within 24 hours.

antibodies in an 8-color staining protocol, detailed in table 1.

CD34 (8G12) BDB

CD14 (M5E2) BD Pharmingen

> CD24 (ALB9) Beckman Coulter

allophycocyanin H 7; PB - pacific blue; PO - pacific orange

, as shown in Figure 1.

CD73 (AD2) BD Pharmingen

NGFR (C40-1457) BD Pharmingen

CD146 (P1H12) BD Pharmingen

> CD105 (1G2) Beckman Coulter

/CD11b-

characteristics.

Tube 1

Tube 2

Tube 3

Tube 4

Tube

CD13+/CD45-

CD49e (SAM1) Beckman Coulter

CD31 (WM59) BD Pharmingen

> CD15 (HI98) BDB

CD106 (51-10C9) BD Pharmingen

By definition, a stem cell is an undifferentiated cell with the potential ability of self-renewal and the capability of differentiation along different cell lineages (multipotency). MSC can be found on a great variety of adult tissues, where they play an important role in tissue regeneration, such as: bone marrow, adipose tissue, umbilical cord blood, umbilical cord matrix, menstrual blood, endometrium, placenta, dental pulp, skin and thymus, among others (Chamberlain et al., 2007; Ding et al. 2011; Kolf et al., 2007; Martins et al., 2009; Musina et al., 2005; Pittenger et al., 1999).

In addition to their presence in numerous adult tissues, MSC are relatively easy to isolate and have the capability to expand manyfold in culture without lose their stem cell properties. Moreover, when MSC are systemically transplanted, they are able to migrate to sites of injury and promote tissue repair, by producing growth factors or other soluble factors important to tissue regeneration, as well as by undergoing cellular differentiation (Chamberlain et al., 2007, Kolf et al., 2007; Mafi et al., 2011); such features explain the success of MSC transfusion therapy in genetic disorders affecting mesenchymal tissues (Horwitz et al., 2002; Undale et al., 2009). Furthermore, those cells have the ability of suppressing the immune response of a wide variety of immune cells, including T, B and NK lymphocytes, and antigen-presenting cells (Chamberlain et al., 2007; Stagg, 2007), and their importance in patients' clinical outcome has already been proven in severe acute graft-versus-host disease (Remberger et al., 2011; von Bahr et al., 2011). Moreover, the results achieved in animal models of autoimmune diseases are promising and encouraged the beginning of phase I clinical trials in multiple sclerosis (Constantin et al., 2009; Darlington et al., 2011; Siatskas et al., 2009).

#### **2.1 Identification and quantification of bone marrow MSC**

As referred previously, the study of minor cell populations with no known specific cell marker toke great advantage on the development of high-speed multi-parameter flow cytometers. The use of an 8-color FACSCanto II (Becton Dickinson Biosciences, BDB) flow cytometer allowed us to identify MSC in bone marrow, quantify them and further characterize their immunophenotypic profile. We employed a monoclonal antibody panel with a backbone of 3 common markers (CD13, CD45 and CD11b) for the identification of MSC (known to be CD13+CD45-CD11b-) in each tube that, at the same time, permitted the study of the expression of five more proteins on MSC per tube.

MSC are rare in bone marrow, being reported that they represent approximately 0,01% of all nucleated bone marrow cells (Chamberlain et al., 2007; Mafi et al., 2011), although is known that their number declines with aging (Caplan, 2007). Our data point to a percentage ranging between 0,01% and 0,03% of all nucleated bone marrow cells (Martins et al., 2009).

#### **2.2 Immunophenotypic characterization of bone marrow MSC**

#### **2.2.1 Flow cytometer quality control, compensation setup strategies and other technical issues**

According to the manufacturer's recommendations, it is done a daily quality control using the Rainbow Beads (BDB). In what concerns to cytometer's compensation setup, it is made once per month by setting up the Rainbow Beads (BDB) values according to the EuroFlow consortium's guidelines and then by doing a general compensation for stable fluorochromes and a specific compensation for each monoclonal antibody conjugated with tandem fluorochromes. Although the compensation is automatic, it is always revised by experienced staff at the end of the process.

In order to detect cellular autofluorescence, a negative control was made for each sample, where the bone marrow sample was only stained for CD45 PO and CD34 PerCPcy5.5.

SSC and FSC light dispersion properties allow a good discrimination between viable and dead cells and the doublets were excluded based on FSC-Area *versus* FSC-Height characteristics.

#### **2.2.2 Material and methods**

458 Flow Cytometry – Recent Perspectives

By definition, a stem cell is an undifferentiated cell with the potential ability of self-renewal and the capability of differentiation along different cell lineages (multipotency). MSC can be found on a great variety of adult tissues, where they play an important role in tissue regeneration, such as: bone marrow, adipose tissue, umbilical cord blood, umbilical cord matrix, menstrual blood, endometrium, placenta, dental pulp, skin and thymus, among others (Chamberlain et al., 2007; Ding et al. 2011; Kolf et al., 2007; Martins et al., 2009;

In addition to their presence in numerous adult tissues, MSC are relatively easy to isolate and have the capability to expand manyfold in culture without lose their stem cell properties. Moreover, when MSC are systemically transplanted, they are able to migrate to sites of injury and promote tissue repair, by producing growth factors or other soluble factors important to tissue regeneration, as well as by undergoing cellular differentiation (Chamberlain et al., 2007, Kolf et al., 2007; Mafi et al., 2011); such features explain the success of MSC transfusion therapy in genetic disorders affecting mesenchymal tissues (Horwitz et al., 2002; Undale et al., 2009). Furthermore, those cells have the ability of suppressing the immune response of a wide variety of immune cells, including T, B and NK lymphocytes, and antigen-presenting cells (Chamberlain et al., 2007; Stagg, 2007), and their importance in patients' clinical outcome has already been proven in severe acute graft-versus-host disease (Remberger et al., 2011; von Bahr et al., 2011). Moreover, the results achieved in animal models of autoimmune diseases are promising and encouraged the beginning of phase I clinical trials in multiple sclerosis (Constantin et al., 2009; Darlington et al., 2011; Siatskas et al., 2009).

As referred previously, the study of minor cell populations with no known specific cell marker toke great advantage on the development of high-speed multi-parameter flow cytometers. The use of an 8-color FACSCanto II (Becton Dickinson Biosciences, BDB) flow cytometer allowed us to identify MSC in bone marrow, quantify them and further characterize their immunophenotypic profile. We employed a monoclonal antibody panel with a backbone of 3 common markers (CD13, CD45 and CD11b) for the identification of MSC (known to be CD13+CD45-CD11b-) in each tube that, at the same time, permitted the

MSC are rare in bone marrow, being reported that they represent approximately 0,01% of all nucleated bone marrow cells (Chamberlain et al., 2007; Mafi et al., 2011), although is known that their number declines with aging (Caplan, 2007). Our data point to a percentage ranging between 0,01% and 0,03% of all nucleated bone marrow cells (Martins et al., 2009).

According to the manufacturer's recommendations, it is done a daily quality control using the Rainbow Beads (BDB). In what concerns to cytometer's compensation setup, it is made once per month by setting up the Rainbow Beads (BDB) values according to the EuroFlow consortium's guidelines and then by doing a general compensation for stable fluorochromes and a specific compensation for each monoclonal antibody conjugated with tandem

**2.2.1 Flow cytometer quality control, compensation setup strategies and other** 

Musina et al., 2005; Pittenger et al., 1999).

**2.1 Identification and quantification of bone marrow MSC** 

study of the expression of five more proteins on MSC per tube.

**2.2 Immunophenotypic characterization of bone marrow MSC** 

**technical issues** 

The immunophenotypic characterization of bone marrow MSC were performed in fresh EDTA-collected bone marrow samples from healthy individuals. After collection the samples were stored at 4 ºC and processed within 24 hours.

Whole bone marrow samples were stained for surface cell markers using a stain-lyse-andthen-wash direct immunofluorescence technique. 200 μl of whole bone marrow were aliquoted in different tubes and stained with the following combinations of monoclonal antibodies in an 8-color staining protocol, detailed in table 1.


Table 1. Panel of monoclonal antibodies used for the bone marrow MSC characterization. FITC - fluorescein isothiocyanate; PE – phycoerythrin; PerCPcy5.5 - peridinin chlorophyll protein cyanine 5.5; PEcy7 - R-phycoerythrin cyanine 7; APC – allophycocyanin; APCH7 allophycocyanin H 7; PB - pacific blue; PO - pacific orange

Data acquisition was performed in a FACSCanto II flow cytometer (BDB), using FACSDiva acquisition software (BDB). The total bone marrow cellularity of the whole sample was acquired (5 x 106 events, minimum) for each tube. Bone marrow MSC were identified as CD13+/CD45-/CD11b- , as shown in Figure 1.

Data analysis was performed using Infinicyt software (Cytognos, Salamanca, Spain).

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 461

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD90 APC

NGFR PE

Fig. 2. Immunophenotypic characteristics of bone marrow MSC (blue). The remaining bone

In what concerns to growth factor receptors, NGFR (nerve growth factor receptor, CD271) is expressed in a wide variety of tissues and, depending on the cell type, signaling through this receptor regulates NF-kB activation, apoptosis, tissue regeneration, immune cell activation, proliferation and cell differentiation (Micera et al., 2007; Rogers et al., 2010). Finally, CD105 (endoglin) is one of the receptors for TGF-β, a growth factor involved in the regulation of development, maintenance and proliferation of MSC (Stagg, 2007), and also

0 50000 150000

SSC-A

CD73 PE

0 1E2 1E3 1E4 1E5

CD146 PE

CD49 FITC

HLA-A, B, C APC

0 50000 150000

SSC-A

CD73 PE

SSC-A

marrow nucleated cells are represented as grey events

known to play an important role in tissue repair.

0 50000 150000

0 1E2 1E3 1E4 1E5

0 50000 150000

0 50000 150000

SSC-A

CD105 PE

0 1E2 1E3 1E4 1E5

SSC-A

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD29 APCH7

CD106 FITC

CD13 PE Cy7

Fig. 1. Identification of bone marrow MSC (blue) present in a whole bone marrow sample, phenotypically characterized as CD13+CD45- CD11b-

#### **2.2.3 Results and discussion**

Bone marrow MSC showed to be uniformly positive to CD13, CD29, CD49e, CD90, CD106, CD146, CD73, NGFR, CD105 and HLA-A, B, C (Figure 1 and Figure 2); and negative to CD24, CD31, CD11b, CD14, CD15, CD34, CD45, CD133 and HLA-DR, which is in agreement with previous studies described in the literature (Chamberlain et al., 2007; Delorme et al., 2008; Ehninger & Trumpp, 2011; Fox et al., 2007; Jones & McGonagle, 2008; Kolf et al., 2007; Martins et al., 2009; Pittenger et al., 1999; Tormin et al., 2011). Based on the expression profile of these markers, bone marrow MSC behave as one sole cell population, as all the studied markers were homogeneously expressed inside the MSC population.

Several studies on adhesion molecules and chemokine receptors expression have been made in order to shed light on MSC migratory and homing ability. CD29 (integrin β1-subunit) and CD106 (vascular cell adhesion molecule 1, VCAM-1) seem to be important in the adhesion of MSC to endothelial cells (Chamberlain et al., 2007; Kolf et al., 2007; Stagg, 2007) and CD29, which when dimerized with CD49e (integrin α5-subunit) forms a receptor that binds to fibronectin and invasin, is likely to promote MSC-extracellular matrix interaction (Gu et al., 2009). CD146 (Muc18) plays an important role in cell-cell and cell-extracellular matrix adhesion and an increased expression of these marker on tumor cells is associated with an increased cell motility and invasiveness/ metastasis capability (Bardin et al., 2001; Zeng et al., 2011). The glycoprotein CD90 (Thy-1) regulates as well cell-cell and cell-extracellular matrix interactions, being involved in adhesion to endothelial cells, migration, metastasis and tissue regeneration (Jurisic et al., 2010; Rege & Hagood, 2006).

The enzyme CD73 is an ecto-5'-nucleotidase that produces extracellular adenosine. In animal tumor models, CD73-generated adenosine inhibits both homing and expansion of T cells via adenosine-receptor signaling. In fact, recent research shows that adenosine suppresses T cell immune response both in activation and effector phases, as well as NK cell immune activity (Wang et al., 2011; Zhang et al., 2010).


CD45 PO

Fig. 1. Identification of bone marrow MSC (blue) present in a whole bone marrow sample,

Bone marrow MSC showed to be uniformly positive to CD13, CD29, CD49e, CD90, CD106, CD146, CD73, NGFR, CD105 and HLA-A, B, C (Figure 1 and Figure 2); and negative to CD24, CD31, CD11b, CD14, CD15, CD34, CD45, CD133 and HLA-DR, which is in agreement with previous studies described in the literature (Chamberlain et al., 2007; Delorme et al., 2008; Ehninger & Trumpp, 2011; Fox et al., 2007; Jones & McGonagle, 2008; Kolf et al., 2007; Martins et al., 2009; Pittenger et al., 1999; Tormin et al., 2011). Based on the expression profile of these markers, bone marrow MSC behave as one sole cell population, as all the

Several studies on adhesion molecules and chemokine receptors expression have been made in order to shed light on MSC migratory and homing ability. CD29 (integrin β1-subunit) and CD106 (vascular cell adhesion molecule 1, VCAM-1) seem to be important in the adhesion of MSC to endothelial cells (Chamberlain et al., 2007; Kolf et al., 2007; Stagg, 2007) and CD29, which when dimerized with CD49e (integrin α5-subunit) forms a receptor that binds to fibronectin and invasin, is likely to promote MSC-extracellular matrix interaction (Gu et al., 2009). CD146 (Muc18) plays an important role in cell-cell and cell-extracellular matrix adhesion and an increased expression of these marker on tumor cells is associated with an increased cell motility and invasiveness/ metastasis capability (Bardin et al., 2001; Zeng et al., 2011). The glycoprotein CD90 (Thy-1) regulates as well cell-cell and cell-extracellular matrix interactions, being involved in adhesion to endothelial cells, migration, metastasis

The enzyme CD73 is an ecto-5'-nucleotidase that produces extracellular adenosine. In animal tumor models, CD73-generated adenosine inhibits both homing and expansion of T cells via adenosine-receptor signaling. In fact, recent research shows that adenosine suppresses T cell immune response both in activation and effector phases, as well as NK cell

studied markers were homogeneously expressed inside the MSC population.

and tissue regeneration (Jurisic et al., 2010; Rege & Hagood, 2006).

immune activity (Wang et al., 2011; Zhang et al., 2010).


CD13 PE Cy7


phenotypically characterized as CD13+CD45-CD11b-


CD13 PE Cy7

CD11b PB

**2.2.3 Results and discussion** 

Fig. 2. Immunophenotypic characteristics of bone marrow MSC (blue). The remaining bone marrow nucleated cells are represented as grey events

In what concerns to growth factor receptors, NGFR (nerve growth factor receptor, CD271) is expressed in a wide variety of tissues and, depending on the cell type, signaling through this receptor regulates NF-kB activation, apoptosis, tissue regeneration, immune cell activation, proliferation and cell differentiation (Micera et al., 2007; Rogers et al., 2010). Finally, CD105 (endoglin) is one of the receptors for TGF-β, a growth factor involved in the regulation of development, maintenance and proliferation of MSC (Stagg, 2007), and also known to play an important role in tissue repair.

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 463

A detailed immunophenotypic description of human bone marrow CD34+ cells was published, few years ago, by Matarraz and colleagues (Matarraz et al., 2008) and Lochem and colleagues (Lochem et al., 2004), along with the frequency of each CD34+ cell

(of total bone marrow) 0,9 ± 0,3 (0,2-1,6)

(within CD34+ cells) 52 ± 12 (19-66)

(within CD34+ cells) 34 ± 7 (15-47)

(within CD34+ cells) 14 ± 10 (1-36)

(within CD34+ cells) 10 ± 7 (0-26)

(within CD34+ cells) 5 ± 2 (0-9)

(within CD34+ cells) 18 ± 8 (1-36)

(within CD34+ cells) 0,7 ± 0,4 (0-1,5)

(within CD34+ cells) 0 ± 0,005 (0-0,02) Table 2. Distribution of the different cell compartments of bone marrow CD34+ HSC. The results are expressed as mean ± standard deviation (range). Adapted from Matarraz et al.

The most immature CD34+ subset can be identified based on CD133 expression (Goussetis et al., 2006; Pastore et al., 2008; Yin et al., 1997). When other markers are concerned, these cell are CD34hi/CD45int/HLA-DRhi/cyMPO-/nTdT-/CD117hi and have intermediate side scatter (SSC) and forward scatter (FSC) light dispersion properties (Matarraz et al., 2008). As previously described by Matarraz and colleagues, the phenotypic profile of CD34+ B cell precursors is CD34int/CD45int/dim/HLA-DRhi/cyMPO-/nTdTint/CD117- and these cells present the lowest SSC and FSC of all CD34+ subpopulations (Lochem et al., 2004; Matarraz et al., 2008); the CD34+ neutrophil precursors present CD34hi/CD45int/dim/HLA-

CD34+ subsets; the CD34+ plasmacytoid dendritic cell precursors are identified based on the expression of CD34+/CD123hi/int/HLA-DRhi; CD34+ monocytic precursors display CD34+/HLA-DRhi/CD64hi/CD45hi/CD117- immunophenotype; basophil precursors are described as being CD34+/CD123int/hi/HLA-DR-/+; and CD34+ mast cell precursors are CD34+/CD117hi/HLA-DR-/int (Matarraz et al., 2008). Finally, CD34+ erythroid precursors are characterized by CD34+/CD36+/CD64-/CD45lo immunophenotype (Matarraz et al., 2008) and by CD105 expression (Buhring et al., 1991; Rokhlin et al., 1995). As a matter of fact, CD105 and TGF-β1 have a pivotal role in the regulation of the differentiation in the

/CD117hi, along with the highest values for SSC and FSC of all

Mean ± Standard deviation

Range

subpopulation in normal hematopoiesis (Matarraz et al. 2008), presented on table 2.

Bone marrow CD34+ HSC compartments

% Bone marrow CD34+ HSC

Immature CD34+ precursor (%)

CD34+ neutrophil precursors (%)

CD34+ monocytic precursors (%)

CD34+ erythroid precursors (%)

CD34+ basophil precursors (%)

CD34+ mast cell precursors (%)

erythroid lineage (Fortunel et al., 2000; Moody et al., 2007).

Leukemia 2008

DRhi/cyMPOint/hi/nTdT-

CD34+ plasmacytoid dendritic cell precursors (%)

CD34+ B cell precursors (%)

Some discrepancies described in the expression of adhesion molecules, chemokine receptors and other proteins, may be the reflex of the microenvironmental differences present in different studies. Although there are a great similitude in the phenotypic profile of MSC isolated from different tissues, differences do exist (Chamberlain et al., 2007; Kolf et al., 2007; Martins et al., 2009). As well as different cultures conditions can also change the MSC phenotype (Chamberlain et al., 2007; Halfon et al., 2011; Stagg, 2007; Tormin et al., 2011). This could be a clue of MSC highly sensitiveness to microenvironment alterations, and their potential to change their protein expression profile could be of great importance in giving an appropriate response to physiological or pathological challenges: by changing their migratory pattern, by initiating an immunomodulatory or immunosuppressive response, by modifying the production and release of soluble factors, or by undergoing cell differentiation.

As a minor bone marrow cell population easy to expand in vitro, it is attractive to characterize the MSC immunophenotype after culture cell expansion. Nevertheless, characterizing these cells directly (without previous culture) enables an analysis closest to their physiological conditions, excluding the phenotypic alterations induced by factors present in the culture medium. Moreover, this direct approach allows an accurate quantification of MSC in bone marrow. Also, this same strategy can be applied to MSC from other tissues.

#### **3. Bone marrow hematopoietic stem cells**

The multipotent hematopoietic stem cell is mainly located in the bone marrow of adult animals and has the ability to differentiate along all hematopoietic cell lineages. A number of studies based on in vitro cell culture, xeno-transplantation of hematopoietic human cells in immunodeficient mice and in pre-immune animal fetuses, were carried out to identify the human hematopoietic stem cell and unveil the hematopoietic precursors hierarchy (Nimer, 2008; Yin et al., 2007), becoming clear that CD34-positive cells were able to differentiate and give rise to all blood cells. There are evidences that, within this heterogeneous population, the more immature CD34+ HSC expresses CD133 and are CD38-negative/dim. It is also known that the CD34+CD133+ subpopulation can arise from the CD133+CD34-CD38 subset (Goussetis et al., 2006; Nimer, 2008; Yin et al., 1997).

#### **3.1 Identification and quantification of the different bone marrow CD34+ HSC cell compartments**

As already referred, CD34-positive cells are an heterogeneous bone marrow cell population, consisting in various cell compartments differing in immunophenotype, size and lineage commitment. The immunophenotypic pattern of each compartment is well described and, with a relatively low number of markers, the majority of those subsets can be accurately and easily identified.

Attending only to the immunophenotypic features, is possible to identify the following bone marrow CD34+ cell subsets by flow cytometry: uncommitted (more immature) precursors, neutrophil precursors, B cell precursors, monocytic precursors, plasmacytoid dendritic cells precursors, erythroid precursors, basophil precursors and mast cell precursors.

Some discrepancies described in the expression of adhesion molecules, chemokine receptors and other proteins, may be the reflex of the microenvironmental differences present in different studies. Although there are a great similitude in the phenotypic profile of MSC isolated from different tissues, differences do exist (Chamberlain et al., 2007; Kolf et al., 2007; Martins et al., 2009). As well as different cultures conditions can also change the MSC phenotype (Chamberlain et al., 2007; Halfon et al., 2011; Stagg, 2007; Tormin et al., 2011). This could be a clue of MSC highly sensitiveness to microenvironment alterations, and their potential to change their protein expression profile could be of great importance in giving an appropriate response to physiological or pathological challenges: by changing their migratory pattern, by initiating an immunomodulatory or immunosuppressive response, by modifying the production and release of soluble factors, or by undergoing cell

As a minor bone marrow cell population easy to expand in vitro, it is attractive to characterize the MSC immunophenotype after culture cell expansion. Nevertheless, characterizing these cells directly (without previous culture) enables an analysis closest to their physiological conditions, excluding the phenotypic alterations induced by factors present in the culture medium. Moreover, this direct approach allows an accurate quantification of MSC in bone marrow. Also, this same strategy can be applied to MSC from

The multipotent hematopoietic stem cell is mainly located in the bone marrow of adult animals and has the ability to differentiate along all hematopoietic cell lineages. A number of studies based on in vitro cell culture, xeno-transplantation of hematopoietic human cells in immunodeficient mice and in pre-immune animal fetuses, were carried out to identify the human hematopoietic stem cell and unveil the hematopoietic precursors hierarchy (Nimer, 2008; Yin et al., 2007), becoming clear that CD34-positive cells were able to differentiate and give rise to all blood cells. There are evidences that, within this heterogeneous population, the more immature CD34+ HSC expresses CD133 and are CD38-negative/dim. It is also known that the CD34+CD133+ subpopulation can arise from the CD133+CD34-CD38-

**3.1 Identification and quantification of the different bone marrow CD34+ HSC cell** 

As already referred, CD34-positive cells are an heterogeneous bone marrow cell population, consisting in various cell compartments differing in immunophenotype, size and lineage commitment. The immunophenotypic pattern of each compartment is well described and, with a relatively low number of markers, the majority of those subsets can be accurately and

Attending only to the immunophenotypic features, is possible to identify the following bone marrow CD34+ cell subsets by flow cytometry: uncommitted (more immature) precursors, neutrophil precursors, B cell precursors, monocytic precursors, plasmacytoid dendritic cells

precursors, erythroid precursors, basophil precursors and mast cell precursors.

differentiation.

other tissues.

**compartments** 

easily identified.

**3. Bone marrow hematopoietic stem cells** 

subset (Goussetis et al., 2006; Nimer, 2008; Yin et al., 1997).

A detailed immunophenotypic description of human bone marrow CD34+ cells was published, few years ago, by Matarraz and colleagues (Matarraz et al., 2008) and Lochem and colleagues (Lochem et al., 2004), along with the frequency of each CD34+ cell subpopulation in normal hematopoiesis (Matarraz et al. 2008), presented on table 2.


Table 2. Distribution of the different cell compartments of bone marrow CD34+ HSC. The results are expressed as mean ± standard deviation (range). Adapted from Matarraz et al. Leukemia 2008

The most immature CD34+ subset can be identified based on CD133 expression (Goussetis et al., 2006; Pastore et al., 2008; Yin et al., 1997). When other markers are concerned, these cell are CD34hi/CD45int/HLA-DRhi/cyMPO-/nTdT-/CD117hi and have intermediate side scatter (SSC) and forward scatter (FSC) light dispersion properties (Matarraz et al., 2008). As previously described by Matarraz and colleagues, the phenotypic profile of CD34+ B cell precursors is CD34int/CD45int/dim/HLA-DRhi/cyMPO-/nTdTint/CD117- and these cells present the lowest SSC and FSC of all CD34+ subpopulations (Lochem et al., 2004; Matarraz et al., 2008); the CD34+ neutrophil precursors present CD34hi/CD45int/dim/HLA-DRhi/cyMPOint/hi/nTdT-/CD117hi, along with the highest values for SSC and FSC of all CD34+ subsets; the CD34+ plasmacytoid dendritic cell precursors are identified based on the expression of CD34+/CD123hi/int/HLA-DRhi; CD34+ monocytic precursors display CD34+/HLA-DRhi/CD64hi/CD45hi/CD117- immunophenotype; basophil precursors are described as being CD34+/CD123int/hi/HLA-DR-/+; and CD34+ mast cell precursors are CD34+/CD117hi/HLA-DR-/int (Matarraz et al., 2008). Finally, CD34+ erythroid precursors are characterized by CD34+/CD36+/CD64-/CD45lo immunophenotype (Matarraz et al., 2008) and by CD105 expression (Buhring et al., 1991; Rokhlin et al., 1995). As a matter of fact, CD105 and TGF-β1 have a pivotal role in the regulation of the differentiation in the erythroid lineage (Fortunel et al., 2000; Moody et al., 2007).

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 465

/CD34hi/HLA-DRhi/CD117hi/FSCint/SSCint/CD123-

SSC-A

CD117 PE Cy7

CD133 APC

bone marrow CD34+ cell compartments are presented in grey

CD35 FITC CD123 PE

2. Bone marrow CD34+ erythroid precursors

FSC-A CD44 PB CD45 PO

CD34 PerCP Cy5.5 HLA-DR APCH7 HLA-DR APCH7

Fig. 3. Uncommitted bone marrow CD34+ HSC (red) immunophenotype. The remaining

characterized by a dim expression of CD34, CD45 and CD44 (Figure 4).

Both CD34+ erythroid precursors and monocytic precursors express CD35. The two CD34+ subpopulations can be distinguished in this protocol by the expression of CD117 and HLA-DR. The erythroid precursors are CD117+/HLA-DRint and the monocytic precursors are CD117dim/-/HLA-DRhi. Moreover, the erythroid precursors are

CD35-

SSC-A

SSC-A

CD123 PE

protocol.

other important phenotypic characteristics have to be taken into account:

immunophenotype of this compartment considering all the markers used in this

. Figure 3 presents a detailed

SSC-A

CD123 PE

#### **3.2 A single-tube protocol to identify the different bone marrow CD34+ HSC compartments**

Recently, we developed an 8-color single-tube protocol to identify the different bone marrow CD34+ HSC subsets by flow cytometry.

The single-tube protocol we propose here was constructed to allow an accurate, quick and easy identification and quantification of those cellular compartments. Attending to the monoclonal antibodies and fluorochrome-conjugation available on the market and to compensation issues, and based on our experience and knowledge on the hematopoietic maturation dynamics, we elected the best markers to identify with precision the cell populations of interest.

#### **3.2.1 Material and methods**

The immunophenotypic characterization of bone marrow CD34+ precursors were performed in fresh EDTA-collected bone marrow samples from healthy individuals. After collection, the samples were stored at 4 ºC and processed within 24 hours. The quality control and compensation strategies are described in detail in section 2.2.1.

A stain-lyse-and-then-wash direct immunofluorescence protocol was used, and the monoclonal antibodies were combined as presented on table 3.


FITC - fluorescein isothiocyanate; PE – phycoerythrin; PerCPcy5.5 - peridinin chlorophyll protein cyanine 5.5; PEcy7 - R-phycoerythrin cyanine 7; APC – allophycocyanin; APCH7 - allophycocyanin H 7; PB - pacific blue; PO - pacific orange.

Table 3. Panel of monoclonal antibodies used for the identification and quantification of the different subpopulations found in bone marrow CD34+ HSC

Data acquisition was performed on a FACSCanto II flow cytometer (BDB), using FACSDiva acquisition software (BDB). In a first step of acquisition, the whole bone marrow cellularity was stored (100.000 events). In a second step, only events within the CD34+ electronic gate were acquired (5.000 to 10.000 CD34+ events).

Data analysis was performed using Infinicyt software (Cytognos, Salamanca, Spain).

#### **3.2.2 How to identify the different CD34+ HSC compartments with the single-tube protocol?**

1. The most immature (uncommitted) compartment of bone marrow C34+ HSC The most immature compartment can be easily identified based on their positivity to CD133 marker (CD133hi). To differentiate this subset from CD34+ neutrophil precursors and CD34+ plasmacytoid dendritic cell precursors, also expressing CD133 (CD133int),

Recently, we developed an 8-color single-tube protocol to identify the different bone

The single-tube protocol we propose here was constructed to allow an accurate, quick and easy identification and quantification of those cellular compartments. Attending to the monoclonal antibodies and fluorochrome-conjugation available on the market and to compensation issues, and based on our experience and knowledge on the hematopoietic maturation dynamics, we elected the best markers to identify with precision the cell

The immunophenotypic characterization of bone marrow CD34+ precursors were performed in fresh EDTA-collected bone marrow samples from healthy individuals. After collection, the samples were stored at 4 ºC and processed within 24 hours. The quality

A stain-lyse-and-then-wash direct immunofluorescence protocol was used, and the

CD117 (PN IM3698) Beckman Coulter

FITC - fluorescein isothiocyanate; PE – phycoerythrin; PerCPcy5.5 - peridinin chlorophyll protein cyanine 5.5; PEcy7 - R-phycoerythrin cyanine 7; APC – allophycocyanin; APCH7 - allophycocyanin H 7;

Table 3. Panel of monoclonal antibodies used for the identification and quantification of the

Data acquisition was performed on a FACSCanto II flow cytometer (BDB), using FACSDiva acquisition software (BDB). In a first step of acquisition, the whole bone marrow cellularity was stored (100.000 events). In a second step, only events within the CD34+ electronic gate

Data analysis was performed using Infinicyt software (Cytognos, Salamanca, Spain).

**3.2.2 How to identify the different CD34+ HSC compartments with the single-tube** 

The most immature compartment can be easily identified based on their positivity to CD133 marker (CD133hi). To differentiate this subset from CD34+ neutrophil precursors and CD34+ plasmacytoid dendritic cell precursors, also expressing CD133 (CD133int),

1. The most immature (uncommitted) compartment of bone marrow C34+ HSC

5.5 PEcy7 APC APCH7 PB PO

HLA-DR (L243) BDB

CD44 (IM7) Biolegend

CD45 (HI30) Invitrogen

CD133 (293C3) Miltenyi Biotec

control and compensation strategies are described in detail in section 2.2.1.

CD34 (8G12) BDB

monoclonal antibodies were combined as presented on table 3.

different subpopulations found in bone marrow CD34+ HSC

were acquired (5.000 to 10.000 CD34+ events).

FITC PE PerCPcy

CD123 (SSDCL Y107D2) Beckman Coulter

**3.2 A single-tube protocol to identify the different bone marrow CD34+ HSC** 

**compartments** 

populations of interest.

Single Tube Protocol

**protocol?** 

**3.2.1 Material and methods** 

CD35 (E11) BDB Pharmingem

PB - pacific blue; PO - pacific orange.

marrow CD34+ HSC subsets by flow cytometry.

other important phenotypic characteristics have to be taken into account: CD35- /CD34hi/HLA-DRhi/CD117hi/FSCint/SSCint/CD123- . Figure 3 presents a detailed immunophenotype of this compartment considering all the markers used in this protocol.

Fig. 3. Uncommitted bone marrow CD34+ HSC (red) immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in grey

2. Bone marrow CD34+ erythroid precursors Both CD34+ erythroid precursors and monocytic precursors express CD35. The two CD34+ subpopulations can be distinguished in this protocol by the expression of CD117 and HLA-DR. The erythroid precursors are CD117+/HLA-DRint and the monocytic precursors are CD117dim/-/HLA-DRhi. Moreover, the erythroid precursors are characterized by a dim expression of CD34, CD45 and CD44 (Figure 4).

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 467

It is worth mentioning that our previous studies with simultaneous staining of CD105 and CD35 proved that the two markers were co-expressed in the same subset of CD34+ bone

0 1E2 1E3 1E4 1E5

Neutrophil precursors show high reactivity to CD44 antigen, as the plasmacytoid dendritic cell precursors (CD44hi), but in the absence of CD123 marker. Other important

Using this single-tube approach, the monocyte precursors are primarily identified by exclusion of all the other myeloid CD34+ precursors. Is noteworthy that a large percentage of monocyte-committed CD34+ precursors express CD35, being discriminated from CD34+ erythroid precursors by their CD117dim/-/HLA-DR+/CD45hi phenotype. Although classically the identification of this CD34+ subset was made focusing on the expression of CD64, this marker seems to be also present on CD34+ plasmacytoid and myeloid dendritic cell precursors. In line with this, CD35 might be a good option to the identification of CD34+ monocyte precursors. The immunophenotype

Even in the absence of an B-cell lineage specific marker, as CD19 or CD79a, CD34+ B cell precursors are clearly identified by the low expression of CD44 and CD45, along

1 According to our experience, CD35 seems to be expressed earlier than CD105 and CD36 on erythroid

committed CD34+ precursors, allowing a more accurate quantification of this subset.

/HLA-

Fig. 5. Expression of CD105 and CD35 in bone marrow erythroid lineage: uncommitted CD34+ cells (red), CD34+erythroid precursors (blue) and CD34- erythroid precursors (grey)

immunophenotypic features of this CD34+ compartment are: CD133int/CD35-

marrow cells and CD35 appears slightly before CD105 (Figure 5)1.

CD35 FITC

DRhi/CD117hi/CD45int/dim/FSChi/SSChi (Figure 6).

3. Bone marrow CD34+ neutrophil precursors

4. Bone marrow CD34+ monocyte precursors

of this population is depicted in Figure 7.

with low light scatter properties (Figure 8).

5. Bone marrow CD34+ B cell precursors

CD105 PE

Fig. 4. Erythroid-committed bone marrow CD34+ precursors (red) immunophenotype. The remaining bone marrow CD34+ cell compartments correspond to the grey events

SSC-A

HLA-DR APCH7

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD34 PerCP Cy5.5 HLA-DR APCH7

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD117 PE Cy7

CD133 APC

0 1E2 1E3 1E4 1E5

CD35 FITC CD123 PE

remaining bone marrow CD34+ cell compartments correspond to the grey events

Fig. 4. Erythroid-committed bone marrow CD34+ precursors (red) immunophenotype. The

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD123 PE

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

FSC-A CD44 PB

0 50000 100000 150000 200000 250000

CD45 PO

SSC-A

SSC-A

SSC-A

CD123 PE

0 1E2 1E3 1E4 1E5

It is worth mentioning that our previous studies with simultaneous staining of CD105 and CD35 proved that the two markers were co-expressed in the same subset of CD34+ bone marrow cells and CD35 appears slightly before CD105 (Figure 5)1.

Fig. 5. Expression of CD105 and CD35 in bone marrow erythroid lineage: uncommitted CD34+ cells (red), CD34+erythroid precursors (blue) and CD34- erythroid precursors (grey)

3. Bone marrow CD34+ neutrophil precursors

Neutrophil precursors show high reactivity to CD44 antigen, as the plasmacytoid dendritic cell precursors (CD44hi), but in the absence of CD123 marker. Other important immunophenotypic features of this CD34+ compartment are: CD133int/CD35- /HLA-DRhi/CD117hi/CD45int/dim/FSChi/SSChi (Figure 6).

4. Bone marrow CD34+ monocyte precursors

Using this single-tube approach, the monocyte precursors are primarily identified by exclusion of all the other myeloid CD34+ precursors. Is noteworthy that a large percentage of monocyte-committed CD34+ precursors express CD35, being discriminated from CD34+ erythroid precursors by their CD117dim/-/HLA-DR+/CD45hi phenotype. Although classically the identification of this CD34+ subset was made focusing on the expression of CD64, this marker seems to be also present on CD34+ plasmacytoid and myeloid dendritic cell precursors. In line with this, CD35 might be a good option to the identification of CD34+ monocyte precursors. The immunophenotype of this population is depicted in Figure 7.

5. Bone marrow CD34+ B cell precursors Even in the absence of an B-cell lineage specific marker, as CD19 or CD79a, CD34+ B cell precursors are clearly identified by the low expression of CD44 and CD45, along with low light scatter properties (Figure 8).

 1 According to our experience, CD35 seems to be expressed earlier than CD105 and CD36 on erythroid committed CD34+ precursors, allowing a more accurate quantification of this subset.

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 469

SSC-A

HLA-DR APCH7

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

FSC-A CD44 PB

CD34 PerCP Cy5.5 HLA-DR APCH7

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD117 PE Cy7

CD133 APC

0 1E2 1E3 1E4 1E5

CD35 FITC CD123 PE

remaining bone marrow CD34+ cell compartments are presented in grey

Fig. 7. Monocytic-committed bone marrow CD34+ precursors (red) immunophenotype. The

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD123 PE

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD45 PO

0 50000 100000 150000 200000 250000

SSC-A

SSC-A

SSC-A

CD123 PE

Fig. 6. Neutrophil-committed bone marrow CD34+ precursors (red) immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in grey

SSC-A

HLA-DR APCH7

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD34 PerCP Cy5.5 HLA-DR APCH7

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD117 PE Cy7

CD133 APC

0 1E2 1E3 1E4 1E5

CD35 FITC CD123 PE

remaining bone marrow CD34+ cell compartments are presented in grey

Fig. 6. Neutrophil-committed bone marrow CD34+ precursors (red) immunophenotype. The

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD123 PE

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

FSC-A CD44 PB

CD45 PO

0 50000 100000 150000 200000 250000

SSC-A

SSC-A

SSC-A

CD123 PE

Fig. 7. Monocytic-committed bone marrow CD34+ precursors (red) immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in grey

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 471

Our protocol allows the identification of basophil precursors using the classical markers and attending to the immunophenotype HLA-DR-/dim/CD123int/hi. Of note, this CD34+ subset presents the lowest expression of CD44 among all bone marrow myeloid CD34+ cells, being easy to differentiate this precursors from all the other myeloid precursors by

The plasmacytoid dendritic cell precursors are identified using the classical markers, as being HLA-DRhi/CD123hi/int. The most immature forms of this precursor express CD133 (CD133int). The immunophenotypic characteristics of this population are

SSC-A

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD123 PE

0 1E2 1E3 1E4 1E5

FSC-A CD44 PB CD45 PO

CD34 PerCP Cy5.5 HLA-DR APCH7 HLA-DR APCH7

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

Fig. 9. Basophil-committed bone marrow CD34+ precursors (red) immunophenotype. The

6. Bone marrow CD34+ basophil precursors

7. Bone marrow CD34+ plasmacytoid dendritic cell precursors

SSC-A

CD117 PE Cy7

0 1E2 1E3 1E4 1E5

CD133 APC

0 1E2 1E3 1E4 1E5

remaining bone marrow CD34+ cell compartments are presented in grey

CD35 FITC CD123 PE

0 1E2 1E3 1E4 1E5

using CD44 marker (Figure 9).

represented on Figure 10.

0 50000 100000 150000 200000 250000

SSC-A

SSC-A

CD123 PE

Fig. 8. B-cell-committed bone marrow CD34+ precursors (red) immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in grey

SSC-A

HLA-DR APCH7

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

FSC-A CD44 PB

CD34 PerCP Cy5.5 HLA-DR APCH7

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD117 PE Cy7

CD133 APC

0 1E2 1E3 1E4 1E5

CD35 FITC CD123 PE

Fig. 8. B-cell-committed bone marrow CD34+ precursors (red) immunophenotype. The

remaining bone marrow CD34+ cell compartments are presented in grey

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

CD123 PE

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD45 PO

0 50000 100000 150000 200000 250000

SSC-A

SSC-A

SSC-A

CD123 PE


Fig. 9. Basophil-committed bone marrow CD34+ precursors (red) immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in grey

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 473

FSC-A CD44 PB

HLA-DR APCH7 HLA-DR APCH7

Fig. 11. Mast cell-committed bone marrow CD34+ precursors (red) immunophenotype. The

The possibility of a multiparameter analysis in a single cell basis conduct to a broader knowledge on the immunophenotypic characteristics of bone marrow CD34+ compartments and how it varies along the differentiation through different hematological cell lineages. Figure 12 depicts the dynamic of the maturation of different bone marrow CD34+ cell

events in grey correspond to remaining whole bone marrow nucleated cells

**3.3 The maturation dynamic of bone marrow CD34+ hematopoietic stem cell** 

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD123 PE 0 1E2 1E3 1E4 1E5

The classical markers for the identification of CD34+ mast cell precursors are included in our protocol, and these cells are CD117hi/HLA-DR-/int. This subset expresses high levels of CD44. Other immunophenotypic characteristics of this subset are illustrated in

SSC-A

CD123 PE

0 1E2 1E3 1E4 1E5

CD35 FITC

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

8. Bone marrow CD34+ mast cell precursors

CD45 PO

0 1E2 1E3 1E4 1E5

0 50000 100000 150000 200000 250000

Figure 11.

SSC-A

SSC-A

CD117 PE Cy7

compartments.

Fig. 10. Plasmacytoid dendritic cell-committed bone marrow CD34+ precursors (red) immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in grey

8. Bone marrow CD34+ mast cell precursors

472 Flow Cytometry – Recent Perspectives

SSC-A

HLA-DR APCH7

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

FSC-A CD44 PB

CD34 PerCP Cy5.5 HLA-DR APCH7

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD117 PE Cy7

CD133 APC

0 1E2 1E3 1E4 1E5

CD35 FITC CD123 PE

immunophenotype. The remaining bone marrow CD34+ cell compartments are presented in

Fig. 10. Plasmacytoid dendritic cell-committed bone marrow CD34+ precursors (red)

0 1E2 1E3 1E4 1E5

CD123 PE

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5 0 1E2 1E3 1E4 1E5

CD45 PO

0 50000 100000 150000 200000 250000

SSC-A

SSC-A

SSC-A

CD123 PE

grey

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

The classical markers for the identification of CD34+ mast cell precursors are included in our protocol, and these cells are CD117hi/HLA-DR-/int. This subset expresses high levels of CD44. Other immunophenotypic characteristics of this subset are illustrated in Figure 11.

Fig. 11. Mast cell-committed bone marrow CD34+ precursors (red) immunophenotype. The events in grey correspond to remaining whole bone marrow nucleated cells

#### **3.3 The maturation dynamic of bone marrow CD34+ hematopoietic stem cell**

The possibility of a multiparameter analysis in a single cell basis conduct to a broader knowledge on the immunophenotypic characteristics of bone marrow CD34+ compartments and how it varies along the differentiation through different hematological cell lineages. Figure 12 depicts the dynamic of the maturation of different bone marrow CD34+ cell compartments.

Immunophenotypic Characterization of Normal Bone Marrow Stem Cells 475

The emergence of high-speed multi-parameter flow cytometers have given an important contribute to unveil the phenotypic characteristics of minor cell populations and/or

Using flow cytometry to characterize bone marrow MSC directly (without in vitro cell culture) represents a great advantage by enabling an analysis closest to the physiologic conditions of the cells, excluding all the phenotypic alterations induced by factors present in the culture medium. Moreover, this direct analysis allows an accurate quantification of these cells in bone marrow. In addition, the strategy used for bone marrow can also be applied in

A broader knowledge about the immunophenotypic characteristics of the different compartments of bone marrow HSC could improve their identification, allow a more accurate quantification of those compartments, as well as shed light on the protein expression patterns in the earliest stages of maturation of each hematological cell lineage. Furthermore, a better knowledge of those protein expression patterns might contribute to the development of new strategies to identify aberrant phenotypes in hematological diseases affecting the more immature bone marrow cells compartments, which can be helpful in the classification of acute leukemias, diagnosis of myelodysplastic syndromes and detection of minimal residual disease. A more extensive understanding of the phenotype of CD34+ hematopoietic stem cells in the different maturational stages could also be useful to monitoring and investigate if different mobilization regimens have the capability of

Here, we presented a simple, quick and economic approach to identify and quantify the

Bardin N, Anfosso F, Massé J, Cramer E, Sabatier F, Le Bivic A, Sampol J & Dignat-George F.

Bühring HJ, Müller CA, Letarte M, Gougos A, Saalmüller A, van Agthoven AJ, Busch FW.

Caplan A. (2007). Adult mesenchymal stem cells for tissue engineering versus regenerative

Chamberlain G, Fox J, Ashton B & Middleton J. (2007). Concise review: mesenchymal stem

Constantin G, Marconi S, Rossi B, Angiari S, Calderan L, Anghileri E, Gini B, Bach D,

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cells: their phenotype, differentiation capacity, immunological features, and potential for homing. *Stem Cells*, Vol.25, No.11 (November 2007), pp. 2739-2749,

Martinello M, Bifari F, Galie M, Turano E, Budui S, Sbarabti A, Krampera M &

MSC from other tissues, allowing their direct quantification and characterization.

mobilizing distinct CD34+ hematopoietic stem cells subpopulations.

different bone marrow CD34+ HSC compartments.

3684, ISSN 0006-4971.

ISSN 0887-6924.

ISSN 1549-4918.

4652.

**4. Conclusion** 

**5. References** 

populations without a known specific cell marker.

Fig. 12. Maturational dynamic of bone marrow CD34+ HSC. Uncommitted CD34+ cells are presented in red, lineage committed CD34+ cells are presented in blue and the lineage committed CD34- cells correspond to grey events

#### **4. Conclusion**

474 Flow Cytometry – Recent Perspectives


A – Neutrophil lineage

HLA-DR APCH7 CD34 PerCP Cy5.5 CD44 PB


B – Erythroid lineage


0 1E2 1E3 1E4 1E5

Fig. 12. Maturational dynamic of bone marrow CD34+ HSC. Uncommitted CD34+ cells are presented in red, lineage committed CD34+ cells are presented in blue and the lineage

SSC-A

SSC-A

CD133 APC -1E2 0 1E2 1E3 1E4 1E5

0 1E3 1E4 1E5

CD117 PE Cy7

CD133 APC

CD133 APC

C – Plasmacytoid dendritic cell lineage

CD123 APC

D – Basophil lineage

0 1E2 1E3 1E4 1E5

HLA-DR APCH7 CD44 PB

committed CD34- cells correspond to grey events



0 1E3 1E4 1E5

CD117 PE Cy7

CD117 PE Cy7

CD117 PE Cy7

CD117 PE Cy7

0 1E3 1E4 1E5

0 1E3 1E4 1E5

0 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5

0 1E2 1E3 1E4 1E5 HLA-DR APCH7


HLA-DR PB

The emergence of high-speed multi-parameter flow cytometers have given an important contribute to unveil the phenotypic characteristics of minor cell populations and/or populations without a known specific cell marker.

Using flow cytometry to characterize bone marrow MSC directly (without in vitro cell culture) represents a great advantage by enabling an analysis closest to the physiologic conditions of the cells, excluding all the phenotypic alterations induced by factors present in the culture medium. Moreover, this direct analysis allows an accurate quantification of these cells in bone marrow. In addition, the strategy used for bone marrow can also be applied in MSC from other tissues, allowing their direct quantification and characterization.

A broader knowledge about the immunophenotypic characteristics of the different compartments of bone marrow HSC could improve their identification, allow a more accurate quantification of those compartments, as well as shed light on the protein expression patterns in the earliest stages of maturation of each hematological cell lineage. Furthermore, a better knowledge of those protein expression patterns might contribute to the development of new strategies to identify aberrant phenotypes in hematological diseases affecting the more immature bone marrow cells compartments, which can be helpful in the classification of acute leukemias, diagnosis of myelodysplastic syndromes and detection of minimal residual disease. A more extensive understanding of the phenotype of CD34+ hematopoietic stem cells in the different maturational stages could also be useful to monitoring and investigate if different mobilization regimens have the capability of mobilizing distinct CD34+ hematopoietic stem cells subpopulations.

Here, we presented a simple, quick and economic approach to identify and quantify the different bone marrow CD34+ HSC compartments.

#### **5. References**


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Pastore D, Mestice A, Perrone T, Gaudio F, Delia M, Albano F, Russo Rossi A, Carluiccio P,

Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,

Rege TA & Hagood JS. (2006). Thy-1 as a regulator of cell-cell and cell-matrix interactions in

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**24** 

*Taiwan* 

**Ethanol Extract of** *Tripterygium wilfordii* 

**Hook. f. Induces G0/G1 Phase Arrest and** 

**Caspase Signaling Pathways** 

*Department of Medical Laboratory Science and Biotechnology,* 

Chung-Jen Chiang et al.\*

*China Medical University, Taichung,* 

**Apoptosis in Human Leukemia HL-60 Cells** 

**Through c-Myc and Mitochondria-Dependent** 

*Tripterygium wilfordii* Hook. f. is a traditional Chinese herb (Murphy, 2006; Qiu et al., 2003). The extract of *Tripterygium wilfordii* Hook. f. has been widely applied to the treatment of immune-related diseases, such as rheumatoid arthritis (RA), nephritis, and systemic lupus erythematosus (SLE) (Chang et al., 1999; Wang et al., 2000). Extracts of *Tripterygium wilfordii*  Hook. f. have been shown to inhibit lymphocyte proliferation induced by mitogentic stimulation *in-vitro* (Wu et al., 2003). Triptolide (PG490, one of the most active components in *Tripterygium wilfordii* Hook. f. extract, possesses immunosuppressive, anti-inflammatory and anti-fertility actions *in vivo* and *in vitro* (Zhao et al., 2005; Leuenroth et al., 2005). Many reports have demonstrated that triptolide has anti-proliferate activity against L1210, U937, K562, HL60, and P388 leukemia cells (Lou et al., 2004; Chan et al., 2001; Wei et al., 1991). However, the cellular and molecular mechanisms underlying mediating *Tripterygium wilfordii* Hook. f.-induced differentiation and/or apoptosis in leukemia cells have not been

Leukemia is a malignant disease characterized by uncontrolled cellular growth and disrupted differentiation of hematopoietic stem cells (Lichtman et al., 2005; O'Hare et al., 2006). Chemotherapy can be effective in certain types of leukemia, but in cases in which it

Jai-Sing Yang1, Yun-Peng Chao2, Li-Jen Lin3 Wen-Wen Huang4, Jing-Gung Chung4, Shu-Fen Peng4,

*3School of Chinese Medicine, China Medical University, Taichung, Taiwan 4Department of Biological Science and Technology, China Medical University, Taichung, Taiwan 5Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan* 

*7Tsuzuki Institute for Traditional Medicine China Medical University, Taichung, Taiwan*

Chi-Cheng Lu5, Jo-Hua Chiang5, Shu-Ren Pai4 and Minoru Tsuzuki6,7 *1Department of Pharmacology, China Medical University, Taichung, Taiwan 2Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan* 

*6Nihon Pharmaceutical University, Saitama, Japan* 

**1. Introduction** 

well studied.

 \*


### **Ethanol Extract of** *Tripterygium wilfordii*  **Hook. f. Induces G0/G1 Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells Through c-Myc and Mitochondria-Dependent Caspase Signaling Pathways**

Chung-Jen Chiang et al.\*

*Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan* 

#### **1. Introduction**

478 Flow Cytometry – Recent Perspectives

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Zeng GF, Cai SX, Wu GJ. (2011). Up-regulation of METCAM/MUC18 promotes motility,

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expression of endoglin on fetal and adult hematopoietic cells in human bone marrow. *J Immunol*, Vol.154, No.9, (May 1995), pp. 4456-4465, ISSN: 0022-1767. Siatskas C, Payne NL, Short MA & Bernard CC. (2010). A consensus statement addressing

mesenchymal stem cell transplantation for multiple sclerosis: it's time! *Stem Cell* 

(2011). CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization. *Blood*, Vol.117, No.19, (May 2011), pp. 5067-77,

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Kearney J & Buck DW. (1997). AC133, a novel marker for human hematopoietic stem and progenitor cells. *Blood*, Vol.90, No.12, (December 1997), pp. 5002-5012,

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*Tripterygium wilfordii* Hook. f. is a traditional Chinese herb (Murphy, 2006; Qiu et al., 2003). The extract of *Tripterygium wilfordii* Hook. f. has been widely applied to the treatment of immune-related diseases, such as rheumatoid arthritis (RA), nephritis, and systemic lupus erythematosus (SLE) (Chang et al., 1999; Wang et al., 2000). Extracts of *Tripterygium wilfordii*  Hook. f. have been shown to inhibit lymphocyte proliferation induced by mitogentic stimulation *in-vitro* (Wu et al., 2003). Triptolide (PG490, one of the most active components in *Tripterygium wilfordii* Hook. f. extract, possesses immunosuppressive, anti-inflammatory and anti-fertility actions *in vivo* and *in vitro* (Zhao et al., 2005; Leuenroth et al., 2005). Many reports have demonstrated that triptolide has anti-proliferate activity against L1210, U937, K562, HL60, and P388 leukemia cells (Lou et al., 2004; Chan et al., 2001; Wei et al., 1991). However, the cellular and molecular mechanisms underlying mediating *Tripterygium wilfordii* Hook. f.-induced differentiation and/or apoptosis in leukemia cells have not been well studied.

Leukemia is a malignant disease characterized by uncontrolled cellular growth and disrupted differentiation of hematopoietic stem cells (Lichtman et al., 2005; O'Hare et al., 2006). Chemotherapy can be effective in certain types of leukemia, but in cases in which it

<sup>\*</sup> Jai-Sing Yang1, Yun-Peng Chao2, Li-Jen Lin3 Wen-Wen Huang4, Jing-Gung Chung4, Shu-Fen Peng4, Chi-Cheng Lu5, Jo-Hua Chiang5, Shu-Ren Pai4 and Minoru Tsuzuki6,7

*<sup>1</sup>Department of Pharmacology, China Medical University, Taichung, Taiwan 2Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan* 

*<sup>3</sup>School of Chinese Medicine, China Medical University, Taichung, Taiwan 4Department of Biological Science and Technology, China Medical University, Taichung, Taiwan 5Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan* 

*<sup>6</sup>Nihon Pharmaceutical University, Saitama, Japan* 

*<sup>7</sup>Tsuzuki Institute for Traditional Medicine China Medical University, Taichung, Taiwan*

Ethanol Extract of *Tripterygium wilfordii* Hook. F. Induces G0/G1

CaliburTM, Becton Dickinson) analysis (Aouacheria et al., 2002).

exposed to Chemiluminescence films (Choi et al., 2003).

measuring OD405 of the released pNA (An et al., 2004).

in ethanol before experiment.

5% CO2 atmosphere.

**2.4 Cell cycle analysis** 

**2.5 Western blotting analysis** 

**2.6 Caspase activities assays** 

2003).

**2.3 Cell culture and viability assay** 

Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells… 481

concentrated by vacuum distillation. The extract was evaporated to dryness and reconstituted

The human promyelocytic leukemia cell line (HL-60) was obtained from the Culture Collection and Research Center (CCRC, Taiwan, R.O.C.), originally from the American Type Culture Collection (ATCC, USA). Cells were cultured in RPMI-1640 culture medium (Gibco/Life Technologies, Taipei, Taiwan) supplemented with 10% heated-inactive fetal bovine serum (Gibco/Life Technologies), 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Gibco/Life Technologies) and incubated at 37°C in humidified

For viability analysis, 2.5 X 105 cells/well were seeded in 24-well culture plates. ETW was added to each well and the plates were incubated at 37°C for 24, 48 and 72 h. Cell viability was estimated by a propidium iodide (PI) incorporation assay and flow cytometry (FACS

Cells were incubated with 50, 100 or 200 μg/mL of ETW for 0, 24 or 48 h. After treatment, cells were washed with phosphate-buffered saline (PBS) twice. The cells were re-suspended in hypotonic PI solution (0.1% sodium citrate, 0.1% Triton X-100, and 50 μg/ml propidium iodide), and then cellular DNA content was determined by flow cytometry (Kamikubo et al.,

Total protein was prepared with protein lysising buffer (PRO-PREPTM protein extraction solution, iNtRON Biotechnology, Seongnam, Gyeonggi-Do, Korea). The concentration of protein was determined by the Bradford method using the Bio-Rad protein assay dye reagent. The lysates containing 40 μg of protein were separated by SDS-PAGE and transferred onto PVDF membrane. Nonspecific binding sites were blocked with 5% non-fat milk in PBST buffer (0.05 % Triton X-100 in PBS) for 1 h. The PVDF membrane was incubated overnight at 4°C with specific primary antibodies against cyclin D1, cyclin E, Bcl-2, and α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After being washed with PBST buffer, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz). Immunoreactive proteins were detected using a Western Blotting Chemiluminescence Reagent Plus kit (NENTM Life Science) and

Cells were collected in lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT) and placed on ice for 10 min. The lysates were centrifuged at 15,800g at 4°C for 10 min. Cell lysates (50 μg of protein) were incubated with caspase -3, -9, and -8 specific substrates (Ac-DEVD-pNA, Ac-LEHD-pNA, and Ac-IETD-pNA) with reaction buffer in a 96-well plate at 37°C for 1 h. The caspase activity was determined by

is not effective additional therapeutic strategies are needed. (Faderl et al., 2005; Frankfurt et al., 2006; ter Bals et al., 2005). Several compounds are capable of inducing the differentiation of leukemia cells into mature cells *in vitro*, and differentiation therapy has been shown to be an effective approach for treating leukemia (Altucci et al., 2004; Altucci et al., 2005; Takahashi et al., 2002). Human promyelocytic leukemia HL-60 cells and mouse monocytic leukemia WEHI-3 cells are commonly used to study various properties of leukemia cell proliferation and differentiation *in vitro* (Lin et al., 2006; Abe et al., 1987). Differentiation of HL-60 is induced into granulocytes by dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA), and into monocytic-like cells by phorbol ester (TPA) and 1,25-dihydroxy-vitamin D3 (Tsiftsoglou et al., 2003). In HL-60 cells, differentiation is induce-specific and is characterized by agents to differentiated is a marked increase in the proportion of G0/G1 cells (Yen et al., 2006), and the modulation of cyclin/CDK (Horie et al., 2004; Wang et al., 1996; Barrera et al., 2004; Pizzimenti et al., 1999; Kumakura et al., 1996).

In hematopoietic cells, apoptosis can be coupled to terminal differentiation of myeloid progenitor (Yazdanparast et al., 2005; Samudio et al., 2005). Cells undergoing apoptosis have observable morphology changes expressed as nuclear condensation, DNA fragmentation, and compact packaging of the cellular debris into apoptotic bodies (Fleischer et al., 2006; Bohm et al., 2006). The delivery and performance of apoptotic signals requires a coordinated cascade of caspase activation and action. The initiator caspases include caspase-8 in Fasinduced apoptosis, and caspase-9, the activation of which is triggered by cytochrome *c* release from mitochondria in response to various stimuli. Those caspases can directly activate downstream effectors of caspase-3, -6, and -7, which cleave death substrates, such as poly(ADP-ribose) polymerase (PARP) (Christophe et al., 2006; Lucken et al., 2005; Lockshin et al., 2005).

In this study, we investigated the cytotoxic effects of ethanol extract of *Tripterygium wilfordii* Hook. f. (ETW) on the promotion of cell cycle arrest and apoptosis in HL-60 cells. Our results indicated that ETW effectively induces both G0/G1 phase arrest and apoptosis of HL-60 cells *in vitro*. The mechanisms governing ETW-induced G0/G1 phase arrest included down regulation of cyclin E, Bcl-2 and Bax, and -triggered apoptosis through caspase-9, caspase-8 and caspase-3-dependent pathways.

#### **2. Materials and methods**

#### **2.1 Chemicals and reagents**

EDTA, Propidium iodide (PI), RNase A, Tris-HCl, Tritox X-100, Tween 20 and Proteinase K were obtained from Sigma Chemical Co. (St.Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), and L-glutamine, penicillin/streptomycin were obtained from Gibco BRL Co. (Grand Island, NY, USA). The caspase-3, caspase-8 and caspase-9 activity assay kits were bought from R&D Systems, Inc. (Minneapolis, MN, USA)

#### **2.2 Ethanol** *Tripterygium wilfordii* **Hook. f. (ETW) extraction**

Dried and powdered plant materials were subjected to continuous ethanol extraction in a Soxhlet extractor with absolute ethanol for 72 h. The ethanol extract was collected and concentrated by vacuum distillation. The extract was evaporated to dryness and reconstituted in ethanol before experiment.

#### **2.3 Cell culture and viability assay**

480 Flow Cytometry – Recent Perspectives

is not effective additional therapeutic strategies are needed. (Faderl et al., 2005; Frankfurt et al., 2006; ter Bals et al., 2005). Several compounds are capable of inducing the differentiation of leukemia cells into mature cells *in vitro*, and differentiation therapy has been shown to be an effective approach for treating leukemia (Altucci et al., 2004; Altucci et al., 2005; Takahashi et al., 2002). Human promyelocytic leukemia HL-60 cells and mouse monocytic leukemia WEHI-3 cells are commonly used to study various properties of leukemia cell proliferation and differentiation *in vitro* (Lin et al., 2006; Abe et al., 1987). Differentiation of HL-60 is induced into granulocytes by dimethyl sulfoxide (DMSO) and all-trans retinoic acid (ATRA), and into monocytic-like cells by phorbol ester (TPA) and 1,25-dihydroxy-vitamin D3 (Tsiftsoglou et al., 2003). In HL-60 cells, differentiation is induce-specific and is characterized by agents to differentiated is a marked increase in the proportion of G0/G1 cells (Yen et al., 2006), and the modulation of cyclin/CDK (Horie et al., 2004; Wang et al., 1996; Barrera et al., 2004; Pizzimenti et al., 1999; Kumakura et al.,

In hematopoietic cells, apoptosis can be coupled to terminal differentiation of myeloid progenitor (Yazdanparast et al., 2005; Samudio et al., 2005). Cells undergoing apoptosis have observable morphology changes expressed as nuclear condensation, DNA fragmentation, and compact packaging of the cellular debris into apoptotic bodies (Fleischer et al., 2006; Bohm et al., 2006). The delivery and performance of apoptotic signals requires a coordinated cascade of caspase activation and action. The initiator caspases include caspase-8 in Fasinduced apoptosis, and caspase-9, the activation of which is triggered by cytochrome *c* release from mitochondria in response to various stimuli. Those caspases can directly activate downstream effectors of caspase-3, -6, and -7, which cleave death substrates, such as poly(ADP-ribose) polymerase (PARP) (Christophe et al., 2006; Lucken et al., 2005; Lockshin

In this study, we investigated the cytotoxic effects of ethanol extract of *Tripterygium wilfordii* Hook. f. (ETW) on the promotion of cell cycle arrest and apoptosis in HL-60 cells. Our results indicated that ETW effectively induces both G0/G1 phase arrest and apoptosis of HL-60 cells *in vitro*. The mechanisms governing ETW-induced G0/G1 phase arrest included down regulation of cyclin E, Bcl-2 and Bax, and -triggered apoptosis through caspase-9,

EDTA, Propidium iodide (PI), RNase A, Tris-HCl, Tritox X-100, Tween 20 and Proteinase K were obtained from Sigma Chemical Co. (St.Louis, MO, USA). RPMI-1640 medium, fetal bovine serum (FBS), and L-glutamine, penicillin/streptomycin were obtained from Gibco BRL Co. (Grand Island, NY, USA). The caspase-3, caspase-8 and caspase-9 activity assay kits

Dried and powdered plant materials were subjected to continuous ethanol extraction in a Soxhlet extractor with absolute ethanol for 72 h. The ethanol extract was collected and

1996).

et al., 2005).

caspase-8 and caspase-3-dependent pathways.

were bought from R&D Systems, Inc. (Minneapolis, MN, USA)

**2.2 Ethanol** *Tripterygium wilfordii* **Hook. f. (ETW) extraction** 

**2. Materials and methods 2.1 Chemicals and reagents** 

The human promyelocytic leukemia cell line (HL-60) was obtained from the Culture Collection and Research Center (CCRC, Taiwan, R.O.C.), originally from the American Type Culture Collection (ATCC, USA). Cells were cultured in RPMI-1640 culture medium (Gibco/Life Technologies, Taipei, Taiwan) supplemented with 10% heated-inactive fetal bovine serum (Gibco/Life Technologies), 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml) (Gibco/Life Technologies) and incubated at 37°C in humidified 5% CO2 atmosphere.

For viability analysis, 2.5 X 105 cells/well were seeded in 24-well culture plates. ETW was added to each well and the plates were incubated at 37°C for 24, 48 and 72 h. Cell viability was estimated by a propidium iodide (PI) incorporation assay and flow cytometry (FACS CaliburTM, Becton Dickinson) analysis (Aouacheria et al., 2002).

#### **2.4 Cell cycle analysis**

Cells were incubated with 50, 100 or 200 μg/mL of ETW for 0, 24 or 48 h. After treatment, cells were washed with phosphate-buffered saline (PBS) twice. The cells were re-suspended in hypotonic PI solution (0.1% sodium citrate, 0.1% Triton X-100, and 50 μg/ml propidium iodide), and then cellular DNA content was determined by flow cytometry (Kamikubo et al., 2003).

#### **2.5 Western blotting analysis**

Total protein was prepared with protein lysising buffer (PRO-PREPTM protein extraction solution, iNtRON Biotechnology, Seongnam, Gyeonggi-Do, Korea). The concentration of protein was determined by the Bradford method using the Bio-Rad protein assay dye reagent. The lysates containing 40 μg of protein were separated by SDS-PAGE and transferred onto PVDF membrane. Nonspecific binding sites were blocked with 5% non-fat milk in PBST buffer (0.05 % Triton X-100 in PBS) for 1 h. The PVDF membrane was incubated overnight at 4°C with specific primary antibodies against cyclin D1, cyclin E, Bcl-2, and α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After being washed with PBST buffer, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (Santa Cruz). Immunoreactive proteins were detected using a Western Blotting Chemiluminescence Reagent Plus kit (NENTM Life Science) and exposed to Chemiluminescence films (Choi et al., 2003).

#### **2.6 Caspase activities assays**

Cells were collected in lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT) and placed on ice for 10 min. The lysates were centrifuged at 15,800g at 4°C for 10 min. Cell lysates (50 μg of protein) were incubated with caspase -3, -9, and -8 specific substrates (Ac-DEVD-pNA, Ac-LEHD-pNA, and Ac-IETD-pNA) with reaction buffer in a 96-well plate at 37°C for 1 h. The caspase activity was determined by measuring OD405 of the released pNA (An et al., 2004).

Ethanol Extract of *Tripterygium wilfordii* Hook. F. Induces G0/G1

represents mean±S.D. from three independent experiments

regulating cyclin D1, cyclin E, Bcl-2 and c-Myc protein expression.

**HL-60 cells** 

**and caspase-3** 

caspase-8.

Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells… 483

Time Sub-G1 G0/G1 S G2/M 0 h 1.19 ± 0.05 52.80 ± 2.64 30.08 ± 1.97 17.12 ± 0.14 24 h 4.58 ± 3.25 77.74 ± 2.59 12.21 ± 2.96 10.05 ± 1.82 48 h 16.68 ± 4.66 81.96 ± 1.35 6.04 ± 1.68 12.00 ± 2.66 Fig. 2. Cell cycle progression on HL-60 cells after treated with ETW. Cells were treated with ETW for the indicated incubation times, then stained for DNA with PI, and analyzed for cell cycle progression or apoptosis by flow cytometry. Cell cycle analysis showed that ETW induced a prominent G0/G1 population arrest and apoptosis in HL-60 cells. Each value

**3.3 Effects of ETW on cyclin D1, cyclin E, Bcl-2 and c-Myc proteins expression in** 

To better understand how ETW induces G0/G1 arrest, we investigated the protein expressions of cyclin D1 and cyclin E. After treatment with 100 μg/ml of ETW, there was a marked increase in protein levels of cyclin D1 and a marked decrease in cyclin E (Fig. 3A and 3B.) We also examined the expression levels of Bcl-2 and c-Myc protein. As shown in Fig. 3C and 3D, Bcl-2 and c-Myc protein levels decreased in HL-60 cells relative to controls. Our results suggest that ETW induces G0/G1 arrest and apoptosis in HL-60 cells by

**3.4 ETW induced apoptosis is mediated by the activations of caspase-9, caspase-8** 

Activation of caspase plays a key role in the induction of apoptosis. We used a fluorogenic enzymatic assay to detect activated caspase-9, caspase-8 and caspase-3 in ETW-treated HL-60 cells. Both caspase-9 and caspase-3 activities increased 24 h after ETW treatment and caspase-8 activities increased 48 h after ETW treatment (Fig. 4). Our results suggest that ETW-induced apoptosis is mediated through the activation of caspase-9, caspase-3 and then

#### **2.7 Statistical analysis**

Results are presented as mean ± S.D. Differences between the different treatment groups, which consisted of matched samples, were assessed by the Student's *t*-test. A p value of less than 0.05 was considered to be significant.

#### **3. Results**

#### **3.1 Effects of ETW on cell viability in HL-60 and WEHI-3 cells**

We treated HL-60 cells with ETW at the concentrations of 0, 50, 100 and 200 μg/ml. The number of viable cells was counted by a PI exclusion method 0, 24 and 48 h later. As shown in Fig. 1, ETW exerted a dose- and time-dependent loss of cell membrane integrity and viability in HL-60 cells.

Fig. 1. Effects of cell viability in ETW treated HL-60 cells. Cells were treated with various concentrations of ETW for indicated duration. Viable cells were measured by PI exclusion and immediately analyzed by flow cytometry. The percentage of cell viability was calculated as a ratio between drug-treated cells and control cells. Each value represents mean±S.D. from three independent experiments

#### **3.2 Effects of ETW on cell cycle progression in HL-60 cells**

To investigate the mechanisms by which ETW induced cytotoxicity effect in HL-60 cells, we cultured cells for various time periods with 100 μg/ml ETW and analyzed DNA content by flow cytometry. Cell cycle analysis showed that ETW induced a prominent G0/G1 population arrest in HL-60 cells (Fig. 2.). In addition, 100 μg/ml of ETW increased the sub-G0/G1 nuclei population in HL-60 cells in a time-dependent manner (Fig. 2.).

Results are presented as mean ± S.D. Differences between the different treatment groups, which consisted of matched samples, were assessed by the Student's *t*-test. A p value of less

We treated HL-60 cells with ETW at the concentrations of 0, 50, 100 and 200 μg/ml. The number of viable cells was counted by a PI exclusion method 0, 24 and 48 h later. As shown in Fig. 1, ETW exerted a dose- and time-dependent loss of cell membrane integrity and

> **0 24 48 Time (hours)**

Fig. 1. Effects of cell viability in ETW treated HL-60 cells. Cells were treated with various concentrations of ETW for indicated duration. Viable cells were measured by PI exclusion

To investigate the mechanisms by which ETW induced cytotoxicity effect in HL-60 cells, we cultured cells for various time periods with 100 μg/ml ETW and analyzed DNA content by flow cytometry. Cell cycle analysis showed that ETW induced a prominent G0/G1 population arrest in HL-60 cells (Fig. 2.). In addition, 100 μg/ml of ETW increased the sub-

and immediately analyzed by flow cytometry. The percentage of cell viability was calculated as a ratio between drug-treated cells and control cells. Each value represents

G0/G1 nuclei population in HL-60 cells in a time-dependent manner (Fig. 2.).

**2.7 Statistical analysis** 

viability in HL-60 cells.

**0**

**20**

**40**

**60**

**Viability (% of control)**

**80**

**100**

**120**

**3. Results** 

than 0.05 was considered to be significant.

**0 ug/ml 50 ug/ml 100 ug/ml 200 ug/ml**

mean±S.D. from three independent experiments

**3.2 Effects of ETW on cell cycle progression in HL-60 cells** 

**3.1 Effects of ETW on cell viability in HL-60 and WEHI-3 cells** 

Fig. 2. Cell cycle progression on HL-60 cells after treated with ETW. Cells were treated with ETW for the indicated incubation times, then stained for DNA with PI, and analyzed for cell cycle progression or apoptosis by flow cytometry. Cell cycle analysis showed that ETW induced a prominent G0/G1 population arrest and apoptosis in HL-60 cells. Each value represents mean±S.D. from three independent experiments

#### **3.3 Effects of ETW on cyclin D1, cyclin E, Bcl-2 and c-Myc proteins expression in HL-60 cells**

To better understand how ETW induces G0/G1 arrest, we investigated the protein expressions of cyclin D1 and cyclin E. After treatment with 100 μg/ml of ETW, there was a marked increase in protein levels of cyclin D1 and a marked decrease in cyclin E (Fig. 3A and 3B.) We also examined the expression levels of Bcl-2 and c-Myc protein. As shown in Fig. 3C and 3D, Bcl-2 and c-Myc protein levels decreased in HL-60 cells relative to controls. Our results suggest that ETW induces G0/G1 arrest and apoptosis in HL-60 cells by regulating cyclin D1, cyclin E, Bcl-2 and c-Myc protein expression.

#### **3.4 ETW induced apoptosis is mediated by the activations of caspase-9, caspase-8 and caspase-3**

Activation of caspase plays a key role in the induction of apoptosis. We used a fluorogenic enzymatic assay to detect activated caspase-9, caspase-8 and caspase-3 in ETW-treated HL-60 cells. Both caspase-9 and caspase-3 activities increased 24 h after ETW treatment and caspase-8 activities increased 48 h after ETW treatment (Fig. 4). Our results suggest that ETW-induced apoptosis is mediated through the activation of caspase-9, caspase-3 and then caspase-8.

Ethanol Extract of *Tripterygium wilfordii* Hook. F. Induces G0/G1

**0.0**

**0.0 0.1 0.2 0.3 0.4 0.5 0.6**

**c-Myc/a-tubulin ratio**

**0.2**

**0.4**

**Bcl-2/a-tubulin ratio**

**0.6**

**0.8**

**1.0**

Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells… 485

\* \*

(c)

**0 24 48 72 (hours)** 

\*

(d)

Fig. 3. Representative Western blotting showing changes on the levels of (A) cyclin D1, (B) cyclin E, (C) Bcl-2 and (D) c-Myc in HL-60 cells after exposure to ETW (100 μg/ml). Cells were treated with ETW for the indicated incubation times then total protein were prepared

\*

− **Bcl-2 (26 Kd)**

− α**-tubulin (55 Kd)**

− **c-Myc (48 Kd)**

− α**-tubulin (55 Kd)**

**0 24 48 72 (hours)** 

**0 24 48 72 Time (h)**

**0 24 48 72 Time (h)**

and determined as described in Materials and Methods

(a)

(b)

− **Cyclin D1 (36 Kd)**

− α**-tubulin (55 Kd)**

− **Cyclin E (45 Kd)**

− α**-tubulin (55 Kd)**

**0 24 48 72 Time (h)**

**0 24 48 72 Time (h)**

\*

(a)

\*

(b)

**0 24 48 72 (hours)** 

**0 24 48 72 (hours)**

\*

**0.0 0.1 0.2 0.3 0.4 0.5 0.6**

**0.0**

**0.2**

**0.4**

**Cyclin E/a-tubulin ratio**

**0.6**

**0.8**

**1.0**

**Cyclin D1/a-tubulin ratio**

(c)

Fig. 3. Representative Western blotting showing changes on the levels of (A) cyclin D1, (B) cyclin E, (C) Bcl-2 and (D) c-Myc in HL-60 cells after exposure to ETW (100 μg/ml). Cells were treated with ETW for the indicated incubation times then total protein were prepared and determined as described in Materials and Methods

(d)

Ethanol Extract of *Tripterygium wilfordii* Hook. F. Induces G0/G1

Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells… 487

al., 2004; Pizzimenti et al., 1999). Thus, it could be suggested that the regulation of cyclin D1 and E as well as CDK2 might anticipate in part the early events in differentiation in ETWtreated HL-60 cells. Our studies found that ETW reduced the level of Bcl-2 and c-Myc in a time-dependent manner. Regulation of the relative levels of Bcl-2 and c-Myc may play an important role in modulating the susceptibility of cells to differentiation (Li et al., 2004; Wu et al., 2002). Previous studies have demonstrated that HL-60 cells exhibited an overexpression of Bcl-2 and c-Myc proto-oncogene and that alteration of cellular oncogenes occur during the differentiation of HL-60 cells (Kumakura et al., 1996). Within myeloid lineage, Bcl-2 is over-expressed in early myeloid precursors but under-expressed or absent in matured myeloid cells and neutrophils (Gazitt et al., 2001; Blagosklonny et al., 1996).

Apoptosis is an evolutionarily conserved process that regulates development and homeostasis, and defects in the mechanisms that regulate cell death are implicated in both tumor genesis and multidrug resistance. Two distinct pathways for apoptosis have been defined, namely the death-receptor pathway and mitochondria pathway (Bohm et al., 2006; Christophe et al., 2006; Lucken et al., 2005; Lockshin et al., 2005). The signal transmitted to the mitochondria pathway causes the release of cytochrome *c* into cytosol. We analyzed apoptosis induction in ETW-treated HL-60 cells by measuring the accumulation of sub-G1 nuclei overtime. We observed the induction of caspase-9 and caspase-8 at 24 h of treatment, and caspase-3 activities at 48 h of treatment before the onset of DNA fragmentation at 72 h treatment by at ETW (Fig. 4). Furthermore, we detected loss of mitochondria membrane potential (ΔΨm) in ETW-treated HL-60 cells and release of mitochondrial cytochrome c to cytosol after 18 h of treatment (data not shown). Recent investigation of triptolide-induced apoptosis of U937 cells has suggested that induced caspase-3 activation and downregulation of the caspase inhibitory protein, XIAP, are involved in this apoptotic process (Choi et al., 2003). Recent reports suggest that DNA damage results in onset of mitochondrial permeability transition, which plays a major role in the apoptotic processes (Choi et al., 2003). A common step in apoptosis involves the loss of mitochondrial membrane potential resulting in increased generation of reactive oxygen species (ROS) from the mitochondrial respiratory chain. Our results suggest that ETW-induced apoptosis is mediated through the loss of mitochondria membrane potential and activation of caspase cascades by activated caspase-9, -8 and caspase-3 in a cytochrome c-dependent manner.

In summary, our results show that ETW induced G0/G1 arrest of HL-60 leukemia cells by regulating the protein expression of cyclin D1, cyclin E, Bcl-2 and c-Myc, and that it induced apoptosis in HL-60 cells by activating caspase-9, caspase-8 and caspase-3. ETW might,

Abe J., Morikawa M., Miyamoto K., Kaiho S., Fukushima M., Miyaura C., Abe E., Suda T.,

Altucci L., Rossin A., Hirsch O., Nebbioso A., Vitoux D., Wilhelm E., Guidez F., De Simone

Nishii Y., 1987. Synthetic analogues of vitamin D3 with an oxygen atom in the side chain skeleton. A trial of the development of vitamin D compounds which exhibit potent differentiation-inducing activity without inducing hypercalcemia. FEBS

M., Schiavone EM., Grimwade D., Zelent A., de The H., Gronemeyer H., 2005. Rexinoid-triggered differentiation and tumor-selective apoptosis of acute myeloid

therefore, be an alternative cancer therapy in treatment of leukemia patients.

**5. References** 

Letters 226, 58-62.

Fig. 4. Effects of ETW induced apoptosis on HL-60 cells by caspases-9, -8 and -3 activities. For caspase activity analysis, aliquots of total cell extracts were incubated with caspases-3, -9 and -8 specific substrates, respectively (Ac-DEVD-pNA, Ac-LEHD-pNA and Ac-IETD-pNA). The release of pNA was measured at 405 nm by a spectrophotometer

#### **4. Discussion**

*Tripterygium wilfordii* Hook. f. (TWHF) is used to treat inflammatory and immune-related diseases. Triptolide, a diterpenoid triepoxide extracted from the TWHF, exerts antitumorigenic actions against leukemia cells. In Differentiation -inducing activity study, some triterpene aglycones and Betulinic acid (pentacyclic triterpene) showed differentiationinducing activity and against human acute promyelocytic leukemia HL-60 cells (Poon, 2004; Umehara et al., 1992). The preclinical laboratory work of identification and testing of potential anti-leukemia agents is designed for three categories: inhibition of cell proliferation, promotion of cell cycle arrest and induction of apoptosis. In the present study, we demonstrated that ETW induces cell cycle arrest and apoptosis in HL-60 cells. Hence, we suggest that ETW is a potent Chinese herb in HL-60 leukemia cells. However, it remains unclear whether ETW effectively induces the elimination of premalignant cells apoptosis *in vivo*.

The effects of ETW on HL-60 cells were associated with a specific disruption of cell cycle events and an induction of G0/G1 arrest. Our results show that ETW led to a loss of cell viability in a dose- and time-dependent manner (Fig. 1). Our study demonstrated that G1 cyclins (cyclin D1 and E) were regulated of HL-60 cells induced by ETW. A recent investigation of leukemia cell differentiation agent-induced differentiation of HL-60 leukemia cells has suggested that TPA to differentiate along the monocyte/macrophage lineage up-regulated of cyclin D1, and all-trans retinoic acid (ATRA) to differentiate along the Granulocyte lineage down-regulated cyclin E expression (Wang et al., 1996; Barrera et

**Caspase-9 activity Caspase-3 activity Caspase-8 activity**

**0 24 48 72 Time (hours)**

Fig. 4. Effects of ETW induced apoptosis on HL-60 cells by caspases-9, -8 and -3 activities. For caspase activity analysis, aliquots of total cell extracts were incubated with caspases-3, -9 and -8 specific substrates, respectively (Ac-DEVD-pNA, Ac-LEHD-pNA and Ac-IETD-pNA).

*Tripterygium wilfordii* Hook. f. (TWHF) is used to treat inflammatory and immune-related diseases. Triptolide, a diterpenoid triepoxide extracted from the TWHF, exerts antitumorigenic actions against leukemia cells. In Differentiation -inducing activity study, some triterpene aglycones and Betulinic acid (pentacyclic triterpene) showed differentiationinducing activity and against human acute promyelocytic leukemia HL-60 cells (Poon, 2004; Umehara et al., 1992). The preclinical laboratory work of identification and testing of potential anti-leukemia agents is designed for three categories: inhibition of cell proliferation, promotion of cell cycle arrest and induction of apoptosis. In the present study, we demonstrated that ETW induces cell cycle arrest and apoptosis in HL-60 cells. Hence, we suggest that ETW is a potent Chinese herb in HL-60 leukemia cells. However, it remains unclear whether ETW effectively induces the elimination of premalignant cells apoptosis *in* 

The effects of ETW on HL-60 cells were associated with a specific disruption of cell cycle events and an induction of G0/G1 arrest. Our results show that ETW led to a loss of cell viability in a dose- and time-dependent manner (Fig. 1). Our study demonstrated that G1 cyclins (cyclin D1 and E) were regulated of HL-60 cells induced by ETW. A recent investigation of leukemia cell differentiation agent-induced differentiation of HL-60 leukemia cells has suggested that TPA to differentiate along the monocyte/macrophage lineage up-regulated of cyclin D1, and all-trans retinoic acid (ATRA) to differentiate along the Granulocyte lineage down-regulated cyclin E expression (Wang et al., 1996; Barrera et

The release of pNA was measured at 405 nm by a spectrophotometer

**0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00**

**4. Discussion** 

*vivo*.

**Caspase activity (OD405 nm)**

al., 2004; Pizzimenti et al., 1999). Thus, it could be suggested that the regulation of cyclin D1 and E as well as CDK2 might anticipate in part the early events in differentiation in ETWtreated HL-60 cells. Our studies found that ETW reduced the level of Bcl-2 and c-Myc in a time-dependent manner. Regulation of the relative levels of Bcl-2 and c-Myc may play an important role in modulating the susceptibility of cells to differentiation (Li et al., 2004; Wu et al., 2002). Previous studies have demonstrated that HL-60 cells exhibited an overexpression of Bcl-2 and c-Myc proto-oncogene and that alteration of cellular oncogenes occur during the differentiation of HL-60 cells (Kumakura et al., 1996). Within myeloid lineage, Bcl-2 is over-expressed in early myeloid precursors but under-expressed or absent in matured myeloid cells and neutrophils (Gazitt et al., 2001; Blagosklonny et al., 1996).

Apoptosis is an evolutionarily conserved process that regulates development and homeostasis, and defects in the mechanisms that regulate cell death are implicated in both tumor genesis and multidrug resistance. Two distinct pathways for apoptosis have been defined, namely the death-receptor pathway and mitochondria pathway (Bohm et al., 2006; Christophe et al., 2006; Lucken et al., 2005; Lockshin et al., 2005). The signal transmitted to the mitochondria pathway causes the release of cytochrome *c* into cytosol. We analyzed apoptosis induction in ETW-treated HL-60 cells by measuring the accumulation of sub-G1 nuclei overtime. We observed the induction of caspase-9 and caspase-8 at 24 h of treatment, and caspase-3 activities at 48 h of treatment before the onset of DNA fragmentation at 72 h treatment by at ETW (Fig. 4). Furthermore, we detected loss of mitochondria membrane potential (ΔΨm) in ETW-treated HL-60 cells and release of mitochondrial cytochrome c to cytosol after 18 h of treatment (data not shown). Recent investigation of triptolide-induced apoptosis of U937 cells has suggested that induced caspase-3 activation and downregulation of the caspase inhibitory protein, XIAP, are involved in this apoptotic process (Choi et al., 2003). Recent reports suggest that DNA damage results in onset of mitochondrial permeability transition, which plays a major role in the apoptotic processes (Choi et al., 2003). A common step in apoptosis involves the loss of mitochondrial membrane potential resulting in increased generation of reactive oxygen species (ROS) from the mitochondrial respiratory chain. Our results suggest that ETW-induced apoptosis is mediated through the loss of mitochondria membrane potential and activation of caspase cascades by activated caspase-9, -8 and caspase-3 in a cytochrome c-dependent manner.

In summary, our results show that ETW induced G0/G1 arrest of HL-60 leukemia cells by regulating the protein expression of cyclin D1, cyclin E, Bcl-2 and c-Myc, and that it induced apoptosis in HL-60 cells by activating caspase-9, caspase-8 and caspase-3. ETW might, therefore, be an alternative cancer therapy in treatment of leukemia patients.

#### **5. References**


Ethanol Extract of *Tripterygium wilfordii* Hook. F. Induces G0/G1

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**25** 

*Greece* 

**Applications of Quantum** 

Dimitrios Kirmizis1, Fani Chatzopoulou2,

*1Medical School, Aristotle University, Thessaloniki,* 

Eleni Gavriilaki2 and Dimitrios Chatzidimitriou2

*2Laboratory of Microbiology, Aristotle University, Thessaloniki,* 

Among several applications of flow cytometry is the identification of cell populations, which is a demanding and often daunting task, given the multitude and the often intercalating pattern of protein expression between different cell types. This complexity nescessitated the use of multicolor flow cytometry, a technique that has been given new perspectives with the emergence of Quandum Dot (QD) technology, which permitted overcoming obstacles, such as limited fluorochrome availability or limited sensitivity of combining multiple organic fluorochromes. The first systematic studies of size-dependent optical properties of semiconductor crystals in colloidal solutions were performed at early 1980s (Henglein, 1982; Brus, 1983). Later, Spanhel et al (1987) performed one of the first core–shell syntheses, a major advance in increasing the quantum yield. Major improvements leading to highly fluorescent QDs were made in the mid-1990s (Hines MA & Guyot-Sionnest P, 1996; Dabbousi RO et al, 1997; Peng et al, 1997). Subsequently, CdSe crystals with silane-modified hydrophilic surfaces were introduced for biological applications (Bruchez et al, 1998; Chan, WCW & Nie S, 1998). Even more recent developments include the encapsulation of CdSe/ZnS core–hell nanocrystals into carboxylated polymer, followed by chemical modification of the surface with long-chain polyethylene glycol (PEG) (Quantum Dot

QDs are inorganic fluorochromes manufactured with the use of semiconductor materials (cadmium selenide for QDs emitting light in the 525- to 655-nm range or cadmium telluride for QDs emitting higher wavelength light) that assemble into nanometer-scale crystals (Chan et al, 2002; Bruchez, 2005). The tiny size of the QD nanocrystals gives these materials unique physical properties which seem tailor-made for multicolour flow cytometry compared to typical semiconductors or other fluorochromes. The most important of them is their broad excitation spectra (Bruchez M, 2005). Actually, QDs can be excited over the entire visual wavelength range as well as far into the ultraviolet. Because of their exceptionally large Stokes shifts (up to 400 nm), QDs can potentially be used for the multicolor detection even by a single laser flow cytometer, whereas organic dyes require

**1. Introduction** 

Corporation, Hayward, CA).

**2. Properties of Quantum Dots** 

**Dots in Flow Cytometry** 


### **Applications of Quantum Dots in Flow Cytometry**

Dimitrios Kirmizis1, Fani Chatzopoulou2, Eleni Gavriilaki2 and Dimitrios Chatzidimitriou2 *1Medical School, Aristotle University, Thessaloniki, 2Laboratory of Microbiology, Aristotle University, Thessaloniki, Greece* 

#### **1. Introduction**

490 Flow Cytometry – Recent Perspectives

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*Tripterygium wilfordii* Hook. f. Drugs in R & D 4, 1-18.

Triptolide, the Principal Active Diterpenoid from the Chinese Medicinal Herb

apoptosis and differentiation in acute myeloid leukemia: identification of isomerspecific antileukemic activities of the pregnadienedione structure. Molecular

differentiation-inducing activities of triterpenes. Chemical & Pharmaceutical

leukemia cells induced by all-trans retinoic acid is enhanced in combination with

Chinese medicinal plant *Tripterygium wilfordii* Hook.: I. prolongation of rat cardiac and renal allograft survival by the PG27 extract and Immunosuppressive Synergy

mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60

stomach cancer cell lines. Zhongguo Yao Li Xue Bao/Acta Pharmacologica Sinica

hTERT, c-myc and bcl-2 during terminal differentiation of HL-60 cells induced by

experimental autoimmune uveoretinitis by down-regulating Th1-type response.

from Dendrostellera lessertii induces differentiation and apoptosis in HL-60 cells.

76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation G0/G1 arrest of HL-60 cells. European Journal of Cell Biology 85,

in rats with severe acute pancreatitis. Hepatobiliary & Pancreatic Diseases

Contraception 36, 335-345.

Cancer Therapeutics 4, 1982-1992.

of Anticancer Therapy 5, 917-929.

cells. Cancer Research 56, 264-267.

Planta Medica 71, 1112-1117.

International 4, 604-608.

Bulletin. 40, 401-405.

12, 406-410.

117-132.

Among several applications of flow cytometry is the identification of cell populations, which is a demanding and often daunting task, given the multitude and the often intercalating pattern of protein expression between different cell types. This complexity nescessitated the use of multicolor flow cytometry, a technique that has been given new perspectives with the emergence of Quandum Dot (QD) technology, which permitted overcoming obstacles, such as limited fluorochrome availability or limited sensitivity of combining multiple organic fluorochromes. The first systematic studies of size-dependent optical properties of semiconductor crystals in colloidal solutions were performed at early 1980s (Henglein, 1982; Brus, 1983). Later, Spanhel et al (1987) performed one of the first core–shell syntheses, a major advance in increasing the quantum yield. Major improvements leading to highly fluorescent QDs were made in the mid-1990s (Hines MA & Guyot-Sionnest P, 1996; Dabbousi RO et al, 1997; Peng et al, 1997). Subsequently, CdSe crystals with silane-modified hydrophilic surfaces were introduced for biological applications (Bruchez et al, 1998; Chan, WCW & Nie S, 1998). Even more recent developments include the encapsulation of CdSe/ZnS core–hell nanocrystals into carboxylated polymer, followed by chemical modification of the surface with long-chain polyethylene glycol (PEG) (Quantum Dot Corporation, Hayward, CA).

#### **2. Properties of Quantum Dots**

QDs are inorganic fluorochromes manufactured with the use of semiconductor materials (cadmium selenide for QDs emitting light in the 525- to 655-nm range or cadmium telluride for QDs emitting higher wavelength light) that assemble into nanometer-scale crystals (Chan et al, 2002; Bruchez, 2005). The tiny size of the QD nanocrystals gives these materials unique physical properties which seem tailor-made for multicolour flow cytometry compared to typical semiconductors or other fluorochromes. The most important of them is their broad excitation spectra (Bruchez M, 2005). Actually, QDs can be excited over the entire visual wavelength range as well as far into the ultraviolet. Because of their exceptionally large Stokes shifts (up to 400 nm), QDs can potentially be used for the multicolor detection even by a single laser flow cytometer, whereas organic dyes require

Applications of Quantum Dots in Flow Cytometry 493

primary determinant of the emission spectrum of each particle is its size so that smaller nanocrystals have different quantum confinement properties than bigger ones, as a result of the fact that the distance jumped by the exciton differs (the band gap is larger), and light is emitted at a different wavelength upon return to resting state. Increasing crystal size (from 2–3 to 10–12 nm) results in shift of the emission maximum from 500 to 800 nm. However, although they fluoresce at different wavelengths, they are excited at the same wavelength, allowing detection of multiple QD colors from just one laser. Thus, QDs with the smallest nanocrystal cores (3 nm) emit light in the blue region of the spectrum, whereas QDs with the largest cores (~6 nm) emit far red light (Bruchez et al, 1998). The most commonly used QDs in multicolor flow cytometry emit light at 525 (referred to as QD525), 545, 565, 585, 605, 655, 705, and 800 nm (Perfetto et al, 2004); their nanocrystal cores range in size from 2 to 6 nm (Biju et al, 2008). Other QDs emitting light at intermediate wavelengths (like 625 nm) are also commercially available. Thus, by 'tuning' the size of the nanocrystal core with various procedures (Peng et al, 1998; Smet et al, 1999), the description of which is beyond the scope of this chapter, QDs of different colors can be produced from the same starting material (*see* 

The core–shell nanocrystals have large extinction coefficients and high quantum yields. These parameters describe the capacity of the system to capture and subsequently rerelease light. Although quantum yields of QD conjugates in aqueous buffers (20–50%) are comparable with those of conventional fluorophores, the excitation efficiency of QD conjugates is much higher, making them about two orders of magnitude more efficient at absorbing excitation light than organic dyes and fluorescent proteins. QDs have a fluorescence lifetime of 20–30 ns about 10 times longer than the background autofluorescence of proteins. Thus, fluorescence from single CdSe crystals has been observed much longer than from other fluorophores, resulting in high turnover rates and a large number of emitted photons (Doose, 2003).

The procedure for conjugation of antibodies to QDs is similar to conjugation of antibodies to PE, with slight variations in the reagents used and ratio of antibodies to fluorescent molecules. Successful conjugation relies on the coupling of malemide groups on the QDs to thiol groups on the antibody. These groups are generated during the initial steps of the procedure, as amine groups on the QDs are activated with a heterobifunctional crosslinker (sulfosuccinimidyl 4-[*N*-maleimidomethyl]cyclohexane-1-carboxylate, sulfo-SMCC) to generate the malemide moieties, and disulfide bonds in the antibody are reduced to thiol groups using dithiothreitol (DTT). Before conjugation, the DTT-reduced antibody is then mixed with two dye-labeled markers, Cyanin-3 (Cy3) and dextran blue, which track the monomeric fraction of antibodies as it passes through purification columns. Activated QDs and reduced antibody are subsequently purified over columns

A number of laser choices are available to excite QDs. Low wavelength ultraviolet (UV) and violet lasers are typically employed, since they induce maximal fluorescence emission. In theory, QD fluorescence arising from UV excitation is greater than that resulting from violet excitation; however, in practice, UV lasers induce much higher autofluorescence of cells, thereby negating the benefit of higher signal intensity. Still, users who rely on UV-excited probes (like DAPI and Hoechst) should note that QDs are compatible with their systems (Telford WG, 2004). Wheremultiplexed analysis of QDs is important, UV or violet excitation systems can be coupled to as many as eight photomultiplier tubes, allowing simultaneous

Fig. 1).

and mixed for conjugation.

multiple lasers for excitation in order to be used in multiplexed analysis (Bruchez M, 2005; Perfetto et al, 2004; Chattopadhyay et al, 2006). The light that the flow cytometer detects is the light which the electrons in QDs, after having been excited (excitons) by light absorption, emit as they return back from their conduction to their valence bands. What differentiates QDs from the typical fluorochromes is the so called "quantum confinement" phenomenon (Andersen et al, 2002), i.e. the phenomenon whereby, in contrast with the typical semiconductor materials where the distances between the bands are intimisimal (continuous), the excitons in QDs jump a discrete distance (known as the band gap) between bands, as a result of the very small size of the QD nanocrystal core. The narrow emission spectra of QDs usually overcomes the need for compensation, a standard process used in organic fluorochromes, which subtracts spillover fluorescence by estimating its magnitude as a fraction of the measured fluorescence in the primary detector (Roederer M, 2001) (*see*  Fig. 1). In practice, it is reported that most QDs can be used simultaneously with only minimal (<10%) compensation between channels (Roederer et al, 2004). Moreover, when QD reagents are used with common fluorochromes excited by 488, 532, or 633 lasers (e.g., fluoroscein isothiacyanate, phycoerythrin [PE], or allophycocyanin), almost no spillover signal from other fluorochromes in the QD channels is found. Thus, in instruments with two or more lasers, QDs can be multiplexed with other fluorochromes to successfully measure even more colors (Chattopadhyay et al, 2007; 2010). In addition, the signals produced can be extremely bright, such as when an ultraviolet (350 nm) or violet (408 nm) laser is used to excite longer wavelength QDs (like QD605 andQD655), because of their high absorbance and low background levels (Hotz CZ, 2005; Wu et al, 2003). Finally, the emission properties of QDs also offer advantages over organic fluorochromes, albeit to a lesser extent.

Whereas organic fluorochromes of different colors come from a wide variety of source materials, each with distinct (and complex) physical, chemical, and biological properties which may not be compatible with each other or with staining conditions, this becomes less of a concern in QDs since QDs of different colors can be synthesized from the same starting materials (Chan et al, 2002), and thus multiplexed analysis is easier. However, the large surface-to-volume ratio in a nanosized crystal (about 50% of all atoms are on the surface) affects the emission of photons. Photochemical oxidation and surface defects in a crystal with no shell may lead to a broad emission and lower quantum yields. Indeed, early QD nanocrystals did not give stable or bright signals, exhibited poor solubility, and could not be attached to biologic probes (Riegler J & Nann T, 2004). These challenges were overcome by coating the QD nanocrystal with various materials such as inorganic zinc-sulfide, which is in turn coated with organic polymers (Bruchez et al, 1998). These organic polymers increase solubility and provide a platform of functional groups (such as amines, NH3) for conjugation to antibodies, streptavidin, and nucleic acids. Because they have similar coatings, QDs of various colors share uniform biophysical properties and a common conjugation procedure. The final QD product is about the size of PE, and can be linked to antibodies using a very similar conjugation chemistry. The shell helps to confine the excitation to the CdSe core and prevent the non-radiative relaxation.

The fluorescent properties of QDs are derived from their nanocrystal cores and not from the overall size of QD, which is actually similar in all QDs as a result of the fact that the cores are coated with various materials as mentioned above. Each QD has its characteristic emission peak, as long as the excitonic energy levels and quantum yields of fluorescence depend on exciton–photon interaction in the crystal and the size of the crystalline core. The

multiple lasers for excitation in order to be used in multiplexed analysis (Bruchez M, 2005; Perfetto et al, 2004; Chattopadhyay et al, 2006). The light that the flow cytometer detects is the light which the electrons in QDs, after having been excited (excitons) by light absorption, emit as they return back from their conduction to their valence bands. What differentiates QDs from the typical fluorochromes is the so called "quantum confinement" phenomenon (Andersen et al, 2002), i.e. the phenomenon whereby, in contrast with the typical semiconductor materials where the distances between the bands are intimisimal (continuous), the excitons in QDs jump a discrete distance (known as the band gap) between bands, as a result of the very small size of the QD nanocrystal core. The narrow emission spectra of QDs usually overcomes the need for compensation, a standard process used in organic fluorochromes, which subtracts spillover fluorescence by estimating its magnitude as a fraction of the measured fluorescence in the primary detector (Roederer M, 2001) (*see*  Fig. 1). In practice, it is reported that most QDs can be used simultaneously with only minimal (<10%) compensation between channels (Roederer et al, 2004). Moreover, when QD reagents are used with common fluorochromes excited by 488, 532, or 633 lasers (e.g., fluoroscein isothiacyanate, phycoerythrin [PE], or allophycocyanin), almost no spillover signal from other fluorochromes in the QD channels is found. Thus, in instruments with two or more lasers, QDs can be multiplexed with other fluorochromes to successfully measure even more colors (Chattopadhyay et al, 2007; 2010). In addition, the signals produced can be extremely bright, such as when an ultraviolet (350 nm) or violet (408 nm) laser is used to excite longer wavelength QDs (like QD605 andQD655), because of their high absorbance and low background levels (Hotz CZ, 2005; Wu et al, 2003). Finally, the emission properties

of QDs also offer advantages over organic fluorochromes, albeit to a lesser extent.

excitation to the CdSe core and prevent the non-radiative relaxation.

Whereas organic fluorochromes of different colors come from a wide variety of source materials, each with distinct (and complex) physical, chemical, and biological properties which may not be compatible with each other or with staining conditions, this becomes less of a concern in QDs since QDs of different colors can be synthesized from the same starting materials (Chan et al, 2002), and thus multiplexed analysis is easier. However, the large surface-to-volume ratio in a nanosized crystal (about 50% of all atoms are on the surface) affects the emission of photons. Photochemical oxidation and surface defects in a crystal with no shell may lead to a broad emission and lower quantum yields. Indeed, early QD nanocrystals did not give stable or bright signals, exhibited poor solubility, and could not be attached to biologic probes (Riegler J & Nann T, 2004). These challenges were overcome by coating the QD nanocrystal with various materials such as inorganic zinc-sulfide, which is in turn coated with organic polymers (Bruchez et al, 1998). These organic polymers increase solubility and provide a platform of functional groups (such as amines, NH3) for conjugation to antibodies, streptavidin, and nucleic acids. Because they have similar coatings, QDs of various colors share uniform biophysical properties and a common conjugation procedure. The final QD product is about the size of PE, and can be linked to antibodies using a very similar conjugation chemistry. The shell helps to confine the

The fluorescent properties of QDs are derived from their nanocrystal cores and not from the overall size of QD, which is actually similar in all QDs as a result of the fact that the cores are coated with various materials as mentioned above. Each QD has its characteristic emission peak, as long as the excitonic energy levels and quantum yields of fluorescence depend on exciton–photon interaction in the crystal and the size of the crystalline core. The primary determinant of the emission spectrum of each particle is its size so that smaller nanocrystals have different quantum confinement properties than bigger ones, as a result of the fact that the distance jumped by the exciton differs (the band gap is larger), and light is emitted at a different wavelength upon return to resting state. Increasing crystal size (from 2–3 to 10–12 nm) results in shift of the emission maximum from 500 to 800 nm. However, although they fluoresce at different wavelengths, they are excited at the same wavelength, allowing detection of multiple QD colors from just one laser. Thus, QDs with the smallest nanocrystal cores (3 nm) emit light in the blue region of the spectrum, whereas QDs with the largest cores (~6 nm) emit far red light (Bruchez et al, 1998). The most commonly used QDs in multicolor flow cytometry emit light at 525 (referred to as QD525), 545, 565, 585, 605, 655, 705, and 800 nm (Perfetto et al, 2004); their nanocrystal cores range in size from 2 to 6 nm (Biju et al, 2008). Other QDs emitting light at intermediate wavelengths (like 625 nm) are also commercially available. Thus, by 'tuning' the size of the nanocrystal core with various procedures (Peng et al, 1998; Smet et al, 1999), the description of which is beyond the scope of this chapter, QDs of different colors can be produced from the same starting material (*see*  Fig. 1).

The core–shell nanocrystals have large extinction coefficients and high quantum yields. These parameters describe the capacity of the system to capture and subsequently rerelease light. Although quantum yields of QD conjugates in aqueous buffers (20–50%) are comparable with those of conventional fluorophores, the excitation efficiency of QD conjugates is much higher, making them about two orders of magnitude more efficient at absorbing excitation light than organic dyes and fluorescent proteins. QDs have a fluorescence lifetime of 20–30 ns about 10 times longer than the background autofluorescence of proteins. Thus, fluorescence from single CdSe crystals has been observed much longer than from other fluorophores, resulting in high turnover rates and a large number of emitted photons (Doose, 2003).

The procedure for conjugation of antibodies to QDs is similar to conjugation of antibodies to PE, with slight variations in the reagents used and ratio of antibodies to fluorescent molecules. Successful conjugation relies on the coupling of malemide groups on the QDs to thiol groups on the antibody. These groups are generated during the initial steps of the procedure, as amine groups on the QDs are activated with a heterobifunctional crosslinker (sulfosuccinimidyl 4-[*N*-maleimidomethyl]cyclohexane-1-carboxylate, sulfo-SMCC) to generate the malemide moieties, and disulfide bonds in the antibody are reduced to thiol groups using dithiothreitol (DTT). Before conjugation, the DTT-reduced antibody is then mixed with two dye-labeled markers, Cyanin-3 (Cy3) and dextran blue, which track the monomeric fraction of antibodies as it passes through purification columns. Activated QDs and reduced antibody are subsequently purified over columns and mixed for conjugation.

A number of laser choices are available to excite QDs. Low wavelength ultraviolet (UV) and violet lasers are typically employed, since they induce maximal fluorescence emission. In theory, QD fluorescence arising from UV excitation is greater than that resulting from violet excitation; however, in practice, UV lasers induce much higher autofluorescence of cells, thereby negating the benefit of higher signal intensity. Still, users who rely on UV-excited probes (like DAPI and Hoechst) should note that QDs are compatible with their systems (Telford WG, 2004). Wheremultiplexed analysis of QDs is important, UV or violet excitation systems can be coupled to as many as eight photomultiplier tubes, allowing simultaneous

Applications of Quantum Dots in Flow Cytometry 495

*Multicolor Flow Cytometry*: The utility of QDs in multicolor flow cytometry has been documented by several studies. Chattopadhyay et al (2006) in their interesting study analyzed the maturity of various antigenspecific T-cell populations using a 17-color staining panel. This panel consisted of 7 QDs and 10 organic fluorochromes, which were measured simultaneously in the same sample. The QD reagents used were conjugates with conventional antibodies (against CD4, CD45RA, and CD57), as well as peptide MHC Class I (pMHCI) multimers designed to detect those antigen-specific T-cells directed against HIV, EBV, and CMV epitopes. By identifying multiple phenotypically distinct subsets within each antigenspecific T-cell population, the remarkable intricacy of T-cell immunity as well as the power of a multiplexed approach was shown. QDs also allowed the reasearchers to measure many antigen-specific populations simultaneously, an important factor when sample

Markers of interest for use in multicolor flow cytometry are assigned to three categories: primary, secondary, and tertiary (Chattopadhyay et al, 2006, 2010; Perfetto et al, 2004; Mahnke YD & Roederer M, 2007). Primary markers are those that are highly expressed on cells, without intermediate fluorescence (i.e., they exhibit on/off expression). Secondary markers alike are expressed brightly and are well-characterized, but can be expressed at intermediate levels, and therefore resolution of dimly staining populations may be important. Thus, the fluorochromes assigned to secondary markers should be those with the less spreading error. Finally, tertiary markers are particularly dim, poorly characterized, or expressed by only a small proportion of cells. For the latter, bright fluorochromes are necessary. In practice, tertiary markers must be considered first. If these markers are particularly dim, they are assigned to fluorochrome channels that receive very little spreading error. QDs are particularly useful in this regard. However, some QDs are dim (QD 525) (Chattopadhyay et al, 2006), and therefore are not suitable for the measurement of dim cell populations. Among QDs, the brightest choices for tertiary markers are QD655, QD605, and QD585, in order of signal intensity. Secondary markers are ideal candidates for conjugation to QDs, especially for slightly dimmer channels, such as QD545, QD565, or QD800, as long as these are often brightly expressed. Finally, primary markers can be assigned to dim channels or to fluorochrome pairs with significant spectral overlap and

*Intracellular staining*: Although QDs are not always compatible with intracellular staining, there have been recent advances in the ability to stain intracellularly with QDs. One approach, designed to avoid steric issues or intracellular degradation, is to target the QD (with or without conjugated antibody) into a cell using enzymes, such as matrix metalloproteinases (Zhang et al, 2006; Tekle et al, 2008), or nuclear or mitochondrial signal peptides (Hoshino et al, 2004). When coupled to antibodies, QDs bound to delivery molecules might allow organelle directed, specific intracellular staining without

*Tetramer production*: In the past, only FITC-, PE-, APC-tetramers were available, which limited panel design because many novel or dimly staining antibodies are only found on these fluorochromes. QDs with SA groups can be used to produce pMHCI multimers (commonly called 'tetramers') (Chattopadhyay et al, 2006), displaying higher valency than PE or APC and, thus, allowing brighter signals and better staining resolution (Chattopadhyay et al, 2008).

**3. Quantum Dot applications** 

availability is limited.

spreading error.

fixation/permeabilization.

measurement of QD545, QD565, QD585, QD605, QD655, QD705, QD800, and/or a violetexcitable organic fluorochrome (Chattopadhyay et al, 2010). To detect QD signals, we employ a filter strategy that first selects light sharply with a dichroic mirror, allowing only light above a certain wavelength to pass (long-pass filter). A second filter (known as a band pass filter) is stationed in front of the PMT, in order to collect a broad band of wavelengths for maximal signal. The light reflected by the first long-pass filter is passed to the next detector where it is queried in a similar fashion.

Fig. 1. Emission spectra of fluorochromes. The colored bars represent the wavelength range of filters used for the detection of each fluorochrome. The grey squares note the overlap of neighboring fluorochromes and signify the need for compensation. A significant overlap of phycoerythrin (PE) and PE-based tandems is apparent, which necessitates high compensation values, whereas their long tails of emission can induce significant spreading error (A). In contrast, such an overlap is avoided in QDs, whose emission spectra are narrow and symmetrical, their sensitivity better and the need for compensation less (B)

#### **3. Quantum Dot applications**

494 Flow Cytometry – Recent Perspectives

measurement of QD545, QD565, QD585, QD605, QD655, QD705, QD800, and/or a violetexcitable organic fluorochrome (Chattopadhyay et al, 2010). To detect QD signals, we employ a filter strategy that first selects light sharply with a dichroic mirror, allowing only light above a certain wavelength to pass (long-pass filter). A second filter (known as a band pass filter) is stationed in front of the PMT, in order to collect a broad band of wavelengths for maximal signal. The light reflected by the first long-pass filter is passed to the next

Fig. 1. Emission spectra of fluorochromes. The colored bars represent the wavelength range of filters used for the detection of each fluorochrome. The grey squares note the overlap of neighboring fluorochromes and signify the need for compensation. A significant overlap of phycoerythrin (PE) and PE-based tandems is apparent, which necessitates high compensation values, whereas their long tails of emission can induce significant spreading error (A). In contrast, such an overlap is avoided in QDs, whose emission spectra are narrow and

symmetrical, their sensitivity better and the need for compensation less (B)

detector where it is queried in a similar fashion.

*Multicolor Flow Cytometry*: The utility of QDs in multicolor flow cytometry has been documented by several studies. Chattopadhyay et al (2006) in their interesting study analyzed the maturity of various antigenspecific T-cell populations using a 17-color staining panel. This panel consisted of 7 QDs and 10 organic fluorochromes, which were measured simultaneously in the same sample. The QD reagents used were conjugates with conventional antibodies (against CD4, CD45RA, and CD57), as well as peptide MHC Class I (pMHCI) multimers designed to detect those antigen-specific T-cells directed against HIV, EBV, and CMV epitopes. By identifying multiple phenotypically distinct subsets within each antigenspecific T-cell population, the remarkable intricacy of T-cell immunity as well as the power of a multiplexed approach was shown. QDs also allowed the reasearchers to measure many antigen-specific populations simultaneously, an important factor when sample availability is limited.

Markers of interest for use in multicolor flow cytometry are assigned to three categories: primary, secondary, and tertiary (Chattopadhyay et al, 2006, 2010; Perfetto et al, 2004; Mahnke YD & Roederer M, 2007). Primary markers are those that are highly expressed on cells, without intermediate fluorescence (i.e., they exhibit on/off expression). Secondary markers alike are expressed brightly and are well-characterized, but can be expressed at intermediate levels, and therefore resolution of dimly staining populations may be important. Thus, the fluorochromes assigned to secondary markers should be those with the less spreading error. Finally, tertiary markers are particularly dim, poorly characterized, or expressed by only a small proportion of cells. For the latter, bright fluorochromes are necessary. In practice, tertiary markers must be considered first. If these markers are particularly dim, they are assigned to fluorochrome channels that receive very little spreading error. QDs are particularly useful in this regard. However, some QDs are dim (QD 525) (Chattopadhyay et al, 2006), and therefore are not suitable for the measurement of dim cell populations. Among QDs, the brightest choices for tertiary markers are QD655, QD605, and QD585, in order of signal intensity. Secondary markers are ideal candidates for conjugation to QDs, especially for slightly dimmer channels, such as QD545, QD565, or QD800, as long as these are often brightly expressed. Finally, primary markers can be assigned to dim channels or to fluorochrome pairs with significant spectral overlap and spreading error.

*Intracellular staining*: Although QDs are not always compatible with intracellular staining, there have been recent advances in the ability to stain intracellularly with QDs. One approach, designed to avoid steric issues or intracellular degradation, is to target the QD (with or without conjugated antibody) into a cell using enzymes, such as matrix metalloproteinases (Zhang et al, 2006; Tekle et al, 2008), or nuclear or mitochondrial signal peptides (Hoshino et al, 2004). When coupled to antibodies, QDs bound to delivery molecules might allow organelle directed, specific intracellular staining without fixation/permeabilization.

*Tetramer production*: In the past, only FITC-, PE-, APC-tetramers were available, which limited panel design because many novel or dimly staining antibodies are only found on these fluorochromes. QDs with SA groups can be used to produce pMHCI multimers (commonly called 'tetramers') (Chattopadhyay et al, 2006), displaying higher valency than PE or APC and, thus, allowing brighter signals and better staining resolution (Chattopadhyay et al, 2008).

Applications of Quantum Dots in Flow Cytometry 497

necessary for covalent attachment of antibodies. Lately, improvements on both nanocrystal core and shell technologies have enabled production of QD conjugates with exceptional brightness and low nonspecific adsorption (Larson DR et al, 2003). Recently, a new generation of QD nanocrystals was introduced with the application of a novel surface chemistry with the use of polymeric shell modified with long-chain, amino-functionalized PEGs. This new generation of QD nanocrystals has low nonspecific binding to cells and can be directly conjugated to antibodies through the introduced amino groups, using

Since QDs are a new technology, their safety and toxicity are still a matter of concern. Although preliminary data from literature employing QDs for *in vivo* imaging of mice suggested that QDs are both safe and nontoxic (Voura et al, 2004; Shiohara et al, 2004; Bruchez, 2005; Gao et al, 2007), recent *in vitro* toxicology studies have questioned this assumption (Shiohara et al, 2004; Male et al, 2008). It appears that QDs coated with organic shells are relatively nontoxic for short incubation periods, but their degradation products (in particular Cd and Se), principally as a result of their oxidation and photolysis, may be toxic. Since QD size, charge, and composition of the outer shell are the main factors determinig oxidation and photolysis, toxicity likely differs by QD color and preparation. Regarding their risk on human health, data suggest that this is rather minimal, as long as QDs cannot enter the skin. However, this might not be true upon inhalation or ingestion as well, where

there seems to be some potential for toxicity (Oberdorster et al, 2005; Hoet et al, 2004)

Although specific applications for QDs are still emerging, the basic technology has matured to the point that it can be relatively easily employed in multicolour flow cytometry. Unfortunately, just as applications for QDs are still nascent, so too is the commercial market for QD reagents. Therefore, to maximize the utility of QDs researchers must turn to in-house conjugations. Once implemented, the powerful potential of QD technology becomes evident. The remarkable spectral properties of QDs allow easy multiplexing, and therefore more information can be acquired from fewer samples. These properties make QDs useful in

Andersen KE, Fong C, Pickett W. Quantum confinement in CdSe nanocrystallites. *J Non-*

Bentzen EL, Tomlinson ID, Mason J, Gresch P,Warnement MR, et al. Surface modification to

Biju V, Itoh T, Anas A, Sujith A, Ishikawa M. Semiconductor quantum dots and metal

Brus, L. E. (1983) A simple model for the ionization potential, electron affinity, and aqueous redox potentials of small semiconductor crystallites*. J. Chem. Phys*. 79, 5566–5571. Bruchez, M., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A. P. (1998) Semiconductor

nanocrystals as fluorescent biological labels. *Science* 281, 2013–2016.

reduce nonspecific binding of quantum dots in live cell assays. *Bioconjug Chem* 

nanoparticles: syntheses, optical properties, and biological applications. *Anal* 

bisfunctional cross-linkers.

**5. Conclusion** 

**6. References** 

studying complex biologic systems.

2005, 16:1488–1494.

*Cryst Solids* 2008, 2002:1105–1110.

*Bioanal Chem* 2008, 391:2469–2495.

*Pathogen detection*: Efforts to detect whole pathogens have been considerably more successful with the introduction of QDs. When applied to a mixture of pathogenic and harmless *Escherichia coli* strains, QDs conjugated to antibodies against *E. coli* can detect one pathogenic bacterium among 99 harmless ones (Hahn et al, 2008). These detection limits are comparable to current assays that use FITC, but QDs are 10-fold brighter and give more accurate results.

*Fluorescence resonance energy transfer (FRET) assays*: Another interesting potential application of QDs is for new QD-based FRET assays. In particular, recent studies have reported on efforts to achieve FRET with QDs as the donor or acceptor fluorochrome (Willard DM & VanOrden A, 2003). This might not be so difficult to do, since QDs may be available in a wide variety of colors but share similar biochemistry and, thus, it is easy to find an acceptor dye that emits fluorescence at the desired wavelength. Furthermore, signal from the acceptor and the donor are well discrete and easily recognised, because of their narrow emission spectra. Similarly, donors and acceptors can be chosen such that spectral overlap is minimized; this reduces background emission and increases sensitivity. These advantages are not available in traditional FRET assays using organic fluorochromes.

#### **4. Caveats, safety & toxicity**

Although QDs are emerging as useful tools in multicolor flow cytometry, they are not fully characterized and occasionally exhibit peculiar properties. As mentioned above, not all antibodies will successfully conjugate to QDs. In particular, markers for intracellular flow cytometry (e.g., reagents for intracellular cytokine detection) have been problematic to conjugate in our facility, owing in part to the presence of excessive quantities of unconjugated QDs, to limited access to intracellular compartments due to QD size-related steric problems, to uneven dispersion of QDs throughout the intracellular environment, or to high sensitivity of QDs to chemicals used in the fixation and permeabilization process associated with intracellular staining (Riegler J & Nann T, 2004; Jaiswal et al, 2004b; Tekle et al, 2008). Variation within the QDs themselves occasionally might also be considerable, due to difficulties in the control of their production process. Thus, subtle differences in incubation time or injection of precursor solutions can cause differences in size distribution, shape, and surface defects among QDs (Dabbousi et al, 1997). These can potentially impact basic properties like fluorescence. As a rule of thumb, when using QDs in multicolor flow cytometry it might be useful to engage compensation controls using exactly the same reagent as the experimental panel. Another matter of potential concern with QDs is storage method and stability, as long as QDs are prone to form aggregates or precipitate out of solution, albeit the organic coating surrounding QDs has significantly improved solubility (Jaiswal J & Simon S, 2004) and any precipitation does not actually result in loss of activity, nor does it affect staining patterns (since these aggregates stain very brightly in all channels and are easily gated out of analyses). Manufacturers typically recommend storage in glass vials or in specially coated, non-adherent plastic tubes, since in standard microcentrifuge tubes, QDs may bind plastic, precipitate, and lose activity, especially at low volumes.

Two important obstacles to biological applications of commercially available QDs until recently are low quantum yields in aqueous buffers and strong aggregation of conjugates, both determined by the surface chemistry. For the use of QDs as antibody labeled probes, their outer layer must insulate the CdSe/ZnS core structure from the aqueous environment, prevent the nonspecific adsorption of QDs to cells, as well as provide the functional groups

*Pathogen detection*: Efforts to detect whole pathogens have been considerably more successful with the introduction of QDs. When applied to a mixture of pathogenic and harmless *Escherichia coli* strains, QDs conjugated to antibodies against *E. coli* can detect one pathogenic bacterium among 99 harmless ones (Hahn et al, 2008). These detection limits are comparable to current assays that use FITC, but QDs are 10-fold brighter and give more accurate results. *Fluorescence resonance energy transfer (FRET) assays*: Another interesting potential application of QDs is for new QD-based FRET assays. In particular, recent studies have reported on efforts to achieve FRET with QDs as the donor or acceptor fluorochrome (Willard DM & VanOrden A, 2003). This might not be so difficult to do, since QDs may be available in a wide variety of colors but share similar biochemistry and, thus, it is easy to find an acceptor dye that emits fluorescence at the desired wavelength. Furthermore, signal from the acceptor and the donor are well discrete and easily recognised, because of their narrow emission spectra. Similarly, donors and acceptors can be chosen such that spectral overlap is minimized; this reduces background emission and increases sensitivity. These advantages

Although QDs are emerging as useful tools in multicolor flow cytometry, they are not fully characterized and occasionally exhibit peculiar properties. As mentioned above, not all antibodies will successfully conjugate to QDs. In particular, markers for intracellular flow cytometry (e.g., reagents for intracellular cytokine detection) have been problematic to conjugate in our facility, owing in part to the presence of excessive quantities of unconjugated QDs, to limited access to intracellular compartments due to QD size-related steric problems, to uneven dispersion of QDs throughout the intracellular environment, or to high sensitivity of QDs to chemicals used in the fixation and permeabilization process associated with intracellular staining (Riegler J & Nann T, 2004; Jaiswal et al, 2004b; Tekle et al, 2008). Variation within the QDs themselves occasionally might also be considerable, due to difficulties in the control of their production process. Thus, subtle differences in incubation time or injection of precursor solutions can cause differences in size distribution, shape, and surface defects among QDs (Dabbousi et al, 1997). These can potentially impact basic properties like fluorescence. As a rule of thumb, when using QDs in multicolor flow cytometry it might be useful to engage compensation controls using exactly the same reagent as the experimental panel. Another matter of potential concern with QDs is storage method and stability, as long as QDs are prone to form aggregates or precipitate out of solution, albeit the organic coating surrounding QDs has significantly improved solubility (Jaiswal J & Simon S, 2004) and any precipitation does not actually result in loss of activity, nor does it affect staining patterns (since these aggregates stain very brightly in all channels and are easily gated out of analyses). Manufacturers typically recommend storage in glass vials or in specially coated, non-adherent plastic tubes, since in standard microcentrifuge

tubes, QDs may bind plastic, precipitate, and lose activity, especially at low volumes.

Two important obstacles to biological applications of commercially available QDs until recently are low quantum yields in aqueous buffers and strong aggregation of conjugates, both determined by the surface chemistry. For the use of QDs as antibody labeled probes, their outer layer must insulate the CdSe/ZnS core structure from the aqueous environment, prevent the nonspecific adsorption of QDs to cells, as well as provide the functional groups

are not available in traditional FRET assays using organic fluorochromes.

**4. Caveats, safety & toxicity** 

necessary for covalent attachment of antibodies. Lately, improvements on both nanocrystal core and shell technologies have enabled production of QD conjugates with exceptional brightness and low nonspecific adsorption (Larson DR et al, 2003). Recently, a new generation of QD nanocrystals was introduced with the application of a novel surface chemistry with the use of polymeric shell modified with long-chain, amino-functionalized PEGs. This new generation of QD nanocrystals has low nonspecific binding to cells and can be directly conjugated to antibodies through the introduced amino groups, using bisfunctional cross-linkers.

Since QDs are a new technology, their safety and toxicity are still a matter of concern. Although preliminary data from literature employing QDs for *in vivo* imaging of mice suggested that QDs are both safe and nontoxic (Voura et al, 2004; Shiohara et al, 2004; Bruchez, 2005; Gao et al, 2007), recent *in vitro* toxicology studies have questioned this assumption (Shiohara et al, 2004; Male et al, 2008). It appears that QDs coated with organic shells are relatively nontoxic for short incubation periods, but their degradation products (in particular Cd and Se), principally as a result of their oxidation and photolysis, may be toxic. Since QD size, charge, and composition of the outer shell are the main factors determinig oxidation and photolysis, toxicity likely differs by QD color and preparation. Regarding their risk on human health, data suggest that this is rather minimal, as long as QDs cannot enter the skin. However, this might not be true upon inhalation or ingestion as well, where there seems to be some potential for toxicity (Oberdorster et al, 2005; Hoet et al, 2004)

#### **5. Conclusion**

Although specific applications for QDs are still emerging, the basic technology has matured to the point that it can be relatively easily employed in multicolour flow cytometry. Unfortunately, just as applications for QDs are still nascent, so too is the commercial market for QD reagents. Therefore, to maximize the utility of QDs researchers must turn to in-house conjugations. Once implemented, the powerful potential of QD technology becomes evident. The remarkable spectral properties of QDs allow easy multiplexing, and therefore more information can be acquired from fewer samples. These properties make QDs useful in studying complex biologic systems.

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### *Edited by Ingrid Schmid*

"Flow Cytometry - Recent Perspectives" is a compendium of comprehensive reviews and original scientific papers. The contents illustrate the constantly evolving application of flow cytometry to a multitude of scientific fields and technologies as well as its broad use as demonstrated by the international composition of the contributing author group. The book focuses on the utilization of the technology in basic sciences and covers such diverse areas as marine and plant biology, microbiology, immunology, and biotechnology. It is hoped that it will give novices a valuable introduction to the field, but will also provide experienced flow cytometrists with novel insights and a better understanding of the subject.

Photo by PhonlamaiPhoto / iStock

Flow Cytometry - Recent Perspectives

Flow Cytometry

Recent Perspectives

*Edited by Ingrid Schmid*