**5.4 Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay**

After treatment with the compound apoptosis, induction can be detected by TUNEL assay using the DeadEnd apoptosis detection kit (dUTP nick-end labeling

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**Figure 7.** *TUNEL assay.*

*DOI: http://dx.doi.org/10.5772/intechopen.90226*

**5.5 Annexin V staining by flow cytometry**

TUNEL assay) from Promega (Madison, USA). Briefly, the cells will be grown in coverslips and treated with the compound for 24 h. The cells can be washed with phosphate-buffered saline and fix by immersing the slides in 4% paraformaldehyde for 25 min at room temperature, washed twice with PBS for 5 min. The cells will be permeabilized with 0.2% Triton X-100 solution in PBS for 5 min, washed twice in PBS, and then covered with 100 μl of equilibration buffer and kept for 5–10 min. The equilibrated areas will be blotted around with tissue paper, and 100 μl of terminal deoxynucleotidyl transferase (Tdt) reaction mix will be added to the sections on the slide and incubated at 37°C for 60 min inside a humidified chamber for the end labeling reaction to occur. Immersing the slides in 2× sodium chloride-sodium citrate buffer for 15 min terminated the reactions. The slides will be washed thrice by immersing in fresh PBS for 5 min to remove the unincorporated biotinylated nucleotides. The endogenous peroxidase activity was blocked by immersing the slides in 0.3% hydrogen peroxide. After washing, horse radish peroxidase-labeled streptavidin solution was applied, and the slides incubated for 30 min. After incubation, the color will be developed with the peroxidase substrate (hydrogen peroxide) and the stable chromogen (diaminobenzidine). The slides will be mounted and examined in a light microscope. The apoptotic index (AI) can be calculated as follows: AI = (number of apoptotic cells/total number) × 100%. TUNEL can also be combined with annexin V to comprise a more robust assay that is capable of distinguishing apoptosis and necrosis going on in the cells. Since annexin V binding is reported to occur prior to DNA fragmentation, it is capable of detecting necrotic or early apoptotic cells that exhibit a negative response from TUNEL [25]. In KB human oral cancer cells treated with compound for 24 h, apoptotic cells can be seen in green color (**Figure 7**).

In most normal, viable eukaryotic cells, the negatively charged phospholipid phosphatidylserine (PS) is located in the cytosolic leaflet of the plasma membrane lipid bilayer. Annexin V is a 36 KDa phospholipid-binding protein and has a high affinity to PS in the presence of physiological concentrations of calcium (Ca2+). Apoptotic cells which are otherwise undetectable by staining with propidium iodide (PI) can be directly detected through the staining with fluorochrome-conjugated annexin V. Dead cells are stained with both annexin V and PI, whereas viable cells cannot be stained with either annexin V or PI. An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid. Phosphatidyl serine from the cytoplasmic interphase translocated to the extracellular surface. This loss of membrane asymmetry was detected using the binding properties of annexin V to phosphatidyl serine. Annexin V FITC is a sensitive probe for identifying the

**Figure 7.** *TUNEL assay.*

*Cell Growth*

**110**

**(TUNEL) assay**

**Figure 6.**

**Figure 5.**

*color.*

(**Figure 5**).

*Hoechst 33342: (a) non-apoptotic cells in blue color, (b) apoptosis cells in fluorescent blue color.*

concentrations of compound for 24 h. After washing once with PBS, the cells can be stained with 100 μl of a mixture (1:1) of acridine orange-ethidium bromide (4 μg/ ml) solutions. The cells can be immediately washed with PBS and observed under fluorescence microscope at 450–490 nm. The effects of the compound treated on human oral cancer cells (KB) for 24 h can be visualize using fluorescent microscope

*Acridine orange-ethidium bromide dual staining: (a) and (b) apoptotic cells in orange color, live cells in green* 

Chromatin condensation will be assessed by nuclear staining with Hoechst 33342 [24]. Briefly, cells will be seeded in a 96 well plate at a density of 5 × 105

and then treated with different concentrations of compounds for 24 h. After washing once with PBS, the cells will be stained with 100 μl of Hoechst 33342 (10 mg/ ml stock) and incubate at room temperature for 5 min. Stained cells can be imaged by fluorescence microscope at 350–460 nm compound induced in KB human oral

**5.4 Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling** 

After treatment with the compound apoptosis, induction can be detected by TUNEL assay using the DeadEnd apoptosis detection kit (dUTP nick-end labeling

cells and treat with different

cells

will be seeded in a 96 well plate at a density of 5 × 105

**5.3 Analysis of cell death by Hoechst 33342 staining**

cancer cells for 24 h, stained with Hoechst 33342 (**Figure 6**).

TUNEL assay) from Promega (Madison, USA). Briefly, the cells will be grown in coverslips and treated with the compound for 24 h. The cells can be washed with phosphate-buffered saline and fix by immersing the slides in 4% paraformaldehyde for 25 min at room temperature, washed twice with PBS for 5 min. The cells will be permeabilized with 0.2% Triton X-100 solution in PBS for 5 min, washed twice in PBS, and then covered with 100 μl of equilibration buffer and kept for 5–10 min. The equilibrated areas will be blotted around with tissue paper, and 100 μl of terminal deoxynucleotidyl transferase (Tdt) reaction mix will be added to the sections on the slide and incubated at 37°C for 60 min inside a humidified chamber for the end labeling reaction to occur. Immersing the slides in 2× sodium chloride-sodium citrate buffer for 15 min terminated the reactions. The slides will be washed thrice by immersing in fresh PBS for 5 min to remove the unincorporated biotinylated nucleotides. The endogenous peroxidase activity was blocked by immersing the slides in 0.3% hydrogen peroxide. After washing, horse radish peroxidase-labeled streptavidin solution was applied, and the slides incubated for 30 min. After incubation, the color will be developed with the peroxidase substrate (hydrogen peroxide) and the stable chromogen (diaminobenzidine). The slides will be mounted and examined in a light microscope. The apoptotic index (AI) can be calculated as follows: AI = (number of apoptotic cells/total number) × 100%. TUNEL can also be combined with annexin V to comprise a more robust assay that is capable of distinguishing apoptosis and necrosis going on in the cells. Since annexin V binding is reported to occur prior to DNA fragmentation, it is capable of detecting necrotic or early apoptotic cells that exhibit a negative response from TUNEL [25]. In KB human oral cancer cells treated with compound for 24 h, apoptotic cells can be seen in green color (**Figure 7**).

### **5.5 Annexin V staining by flow cytometry**

In most normal, viable eukaryotic cells, the negatively charged phospholipid phosphatidylserine (PS) is located in the cytosolic leaflet of the plasma membrane lipid bilayer. Annexin V is a 36 KDa phospholipid-binding protein and has a high affinity to PS in the presence of physiological concentrations of calcium (Ca2+). Apoptotic cells which are otherwise undetectable by staining with propidium iodide (PI) can be directly detected through the staining with fluorochrome-conjugated annexin V. Dead cells are stained with both annexin V and PI, whereas viable cells cannot be stained with either annexin V or PI. An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid. Phosphatidyl serine from the cytoplasmic interphase translocated to the extracellular surface. This loss of membrane asymmetry was detected using the binding properties of annexin V to phosphatidyl serine. Annexin V FITC is a sensitive probe for identifying the

**Figure 8.** *Apoptotic KB human oral cancer cells after treatment with compound showing apoptosis.*

apoptotic cells which binds to negatively charged phospholipid surfaces. Annexin V FITC staining precedes the loss of membrane integrity which accompanies the later stages of cell death resulting from apoptotic or rather necrotic process. Therefore, staining with annexin V FITC is conjugated to vital dye propidium iodide. The assay distinguishes between viable cells (annexin V –ve, PI –ve), early apoptotic cells (annexin +ve PI –ve), late apoptotic cells (annexin +ve PI +ve), and necrotic cells (annexin –ve PI +ve) (**Figure 8**). Annexin V is represented in red. An apoptotic cell stained with annexin V (green) is surrounded by potential phagocytes. The PSR (orange) might bind to exposed PS that is configured in a recognizable "eat-me" form by annexin I (bottom) (**Figure 8**). Alternatively, PSR might bind to a ligand composed of PS and annexin I (upper left) or PSR might bind annexin I, which serves as a bridge between exposed PS on the dying cell and PSR on the phagocyte (upper right) [26].

#### **5.6 Assessment of caspase 3 expression by flow cytometry**

Caspase-3 is a key protease that is activated during apoptosis. Briefly, the cells will be treated with various concentrations of compound. After 24 h of incubation, the cells will be washed twice with cold PBS and prepared for acquisition using FITC conjugated monoclonal active caspase-3 antibody apoptosis detection kit. The cells can be fixed in cytofix solution at a concentration of 1 × 106 cells/0.5 ml. The cells will be fixed in ice for 30 min, resuspended in perm wash buffer containing antibody, and incubated for 30 min at room temperature. Analyses by flow cytometry. 10,000 cells can be acquired, and the results can be interpreted using DIVA software analysis (**Figure 9**).

#### **5.7 Determination of caspase activity by fluorimetry**

The ApoAlert caspase assay plates contain the fluorogenic substrates specific for different caspases immobilized in the wells of a 96-well plate. When cell lysate containing the active caspase is applied to the wells, caspase will cleave its substrate, and a fluorescent product will be released that can be detected with a standard fluorescence plate reader. Caspase assay plates enable the analysis of the apoptotic caspase response. This assay design is ideal for studies involving multiple cell types or multiple cell treatments. These plates are provided in two formats: a single caspase format for studies that focus on a specific caspase or a profiling format for analyzing several different caspases simultaneously (caspase-3, caspase-8, caspase-9, and caspase-2).

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**Figure 9.**

**Figure 10.**

*DOI: http://dx.doi.org/10.5772/intechopen.90226*

*Caspase 3 cleavage of KB human oral cancer cells.*

**5.8 Detection of apoptosis by cell cycle analysis**

*Cell cycle analysis showing KB human oral cancer cells.*

Pro software analysis (**Figure 10**).

**6. Conclusions**

The phase of the cell cycle at which compounds treated cancer cells got arrested

can be determined using a fluorescent-activated cell sorter (FACS—Becton Dickinson). The cells will be grown in tissue culture flasks and treat with different concentrations of compounds. After 24 h of treatment, the cells can be harvested and spun down at 3500 rpm for 7 min. The cells can be then fixed in 70% ethanol for 30 min. After centrifugation, the pellet can be dissolved in PBS, and 5 μl of RNAse A (10 mg/ml) will be added and incubated for 30 min at 37°C. About 10 μg/ ml of propidium iodide will be added, and after 15 min of incubation in the dark, the cells can filtered through 0.75 μm filter and analyzed by flow cytometry. Thirty thousand cells can be acquired, and the results can be interpreted using CellQuest

An ideal compound should possess no toxic effects on normal human lymphocyte but at the same time exhibited cytotoxic activity on tumor cell lines. The time- and dose-dependent cytotoxic effect of the compound can be tested through *Cell-Based Assays in Cancer Research DOI: http://dx.doi.org/10.5772/intechopen.90226*

*Cell Growth*

**Figure 8.**

apoptotic cells which binds to negatively charged phospholipid surfaces. Annexin V FITC staining precedes the loss of membrane integrity which accompanies the later stages of cell death resulting from apoptotic or rather necrotic process. Therefore, staining with annexin V FITC is conjugated to vital dye propidium iodide. The assay distinguishes between viable cells (annexin V –ve, PI –ve), early apoptotic cells (annexin +ve PI –ve), late apoptotic cells (annexin +ve PI +ve), and necrotic cells (annexin –ve PI +ve) (**Figure 8**). Annexin V is represented in red. An apoptotic cell stained with annexin V (green) is surrounded by potential phagocytes. The PSR (orange) might bind to exposed PS that is configured in a recognizable "eat-me" form by annexin I (bottom) (**Figure 8**). Alternatively, PSR might bind to a ligand composed of PS and annexin I (upper left) or PSR might bind annexin I, which serves as a bridge between exposed PS on the dying cell and PSR on the phagocyte (upper right) [26].

*Apoptotic KB human oral cancer cells after treatment with compound showing apoptosis.*

Caspase-3 is a key protease that is activated during apoptosis. Briefly, the cells will be treated with various concentrations of compound. After 24 h of incubation, the cells will be washed twice with cold PBS and prepared for acquisition using FITC conjugated monoclonal active caspase-3 antibody apoptosis detection kit. The

cells will be fixed in ice for 30 min, resuspended in perm wash buffer containing antibody, and incubated for 30 min at room temperature. Analyses by flow cytometry. 10,000 cells can be acquired, and the results can be interpreted using DIVA

The ApoAlert caspase assay plates contain the fluorogenic substrates specific for different caspases immobilized in the wells of a 96-well plate. When cell lysate containing the active caspase is applied to the wells, caspase will cleave its substrate, and a fluorescent product will be released that can be detected with a standard fluorescence plate reader. Caspase assay plates enable the analysis of the apoptotic caspase response. This assay design is ideal for studies involving multiple cell types or multiple cell treatments. These plates are provided in two formats: a single caspase format for studies that focus on a specific caspase or a profiling format for analyzing several different caspases simultaneously (caspase-3, caspase-8, cas-

cells/0.5 ml. The

**5.6 Assessment of caspase 3 expression by flow cytometry**

cells can be fixed in cytofix solution at a concentration of 1 × 106

**5.7 Determination of caspase activity by fluorimetry**

software analysis (**Figure 9**).

pase-9, and caspase-2).

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**Figure 9.** *Caspase 3 cleavage of KB human oral cancer cells.*

**Figure 10.** *Cell cycle analysis showing KB human oral cancer cells.*

#### **5.8 Detection of apoptosis by cell cycle analysis**

The phase of the cell cycle at which compounds treated cancer cells got arrested can be determined using a fluorescent-activated cell sorter (FACS—Becton Dickinson). The cells will be grown in tissue culture flasks and treat with different concentrations of compounds. After 24 h of treatment, the cells can be harvested and spun down at 3500 rpm for 7 min. The cells can be then fixed in 70% ethanol for 30 min. After centrifugation, the pellet can be dissolved in PBS, and 5 μl of RNAse A (10 mg/ml) will be added and incubated for 30 min at 37°C. About 10 μg/ ml of propidium iodide will be added, and after 15 min of incubation in the dark, the cells can filtered through 0.75 μm filter and analyzed by flow cytometry. Thirty thousand cells can be acquired, and the results can be interpreted using CellQuest Pro software analysis (**Figure 10**).

#### **6. Conclusions**

An ideal compound should possess no toxic effects on normal human lymphocyte but at the same time exhibited cytotoxic activity on tumor cell lines. The time- and dose-dependent cytotoxic effect of the compound can be tested through the various viability assays discussed in this chapter. The compound might have the potential to induce programmed cell death in cancer cells and can be confirmed through apoptotic studies. Induction of apoptosis in cancer cells is recognized as an efficient strategy for cancer chemotherapy. Apoptosis also seems to be a reliable marker for the evaluation of potential agents to bring out for cancer prevention. Cell-based assays are useful for the assessment of live cells and apoptotic cells after treatment with therapeutic agents. The efficacy of compounds in vitro testing before entering into the clinical trials helps to bring out potent drugs into the limelight for the treatment of diseases. Cell-based assays are useful for evaluating therapeutic potency of the developing approved drugs and biologics for the clinical management of cancer.
