**3.2 TUNEL method**

The TUNEL assay [35] distinguishes between normal and apoptotic cells based on the amount of FITC-dUTP incorporation into the broken DNA ends in the intact single apoptotic nuclei or apoptotic bodies, whereas the normal or growing cells have almost no DNA breaks and will not be stained. This method accurately reflects the most typical biochemical and morphological features of apoptosis and can used with paraffin-embedded tissue sections, frozen tissue sections, cultured cells, and tissue-separated cells. The method is simple, easy, and sensitive in detecting a very small amount of apoptotic cells.

#### **Figure 3.**

*Methods in measuring apoptosis: (A) lymphocytes stained with EB/AO solution. Triangle, arrow, and arrowhead show viable, early apoptotic and late apoptotic cells, respectively. (B) Transmission electron image of a broken apoptotic lymphocyte. The cell breaks up into apoptotic bodies containing organelles or the condensed nuclear fragments. (C) Scanning electron image of a macrophage phagocytosing the apoptotic lymphocytes (asterisks). (D) A macrophage engulfing an apoptotic lymphocyte L, containing the condensed nuclear fragments. Arrows show phagosomes containing the apoptotic bodies of lymphocytes. (E, F) A macrophage phagocytosing apoptotic cells. (G–J) LC3 fluorescent stain in macrophages phagocytosing the apoptotic lymphocytes. The nuclei were stained with DAPI (G) or PI (H). (K and L) PI staining was used to evaluate the apoptosis.*

The limitations of the TUNEL method are as follows. Firstly, it labels apoptotic cells only in the middle and late stages. Secondly, necrotic cells also contain DNA breaks and are labeled by dUTP, so the assay is not specific enough to distinguish between apoptotic and necrotic cells. Thirdly, cells need to be fixed during the labeling process, which may lead to excessive cell debris or loss of DNA fragments. Lastly, subjective factors are involved when counting the number of the apoptotic cells.

In addition, agarose gel electrophoresis can be used to detect the apoptotic DNA fragmentation into an integer multiple of 180–200 bp [36]. ELISA can be performed using anti-DNA and anti-histone monoclonal antibody to detect nucleosome fragments [37]. Mitochondrial membrane potential measurement can distinguish early apoptosis [38] and provides a nice complement to the TUNNEL method.

#### **3.3 Fluorescent staining for flow cytometry analysis**

Application of annexin V is a widely used method for detecting apoptosis [39]. Annexin V is a Ca2 + −dependent phospholipid binding protein with a molecular weight of 35–36KD, which can bind with phosphatidylserine with high

**43**

dual-label method.

**4. Cell proliferation**

*Cell Growth Measurement*

fluorescence microscopy.

and necrosis can be distinguished.

**3.4 Double or multiple staining**

can distinguish apoptosis from living cells [44].

*DOI: http://dx.doi.org/10.5772/intechopen.86835*

affinity. Annexin V can be labeled with fluorescein isothiocyanate (FITC, PE) or biotin and used as a fluorescent probe to detect apoptosis by flow cytometry or

However, the limitation of both staining methods is the poor sensitivity. It is difficult to detect DNA fragments in early apoptotic cells and easy to miss the detection of apoptotic cells in S phase or G2/M phase. Meanwhile, the PI method is

To distinguish the apoptotic cells, necrotic cells, and living cells, researchers perform flow cytometric analysis after double staining with annexin V and PI. Annexin V-FITC has poor membrane permeability and can specifically bind phosphatidylserine; for live cells, no phosphatidylserine can be detected by annexin V, but during apoptosis, phosphatidylserine is valgus outward to the outer side of the cell membrane for easy detection. The fluorescent dye PI binds to chromatin but does not enter the cytoplasm of live cells. However, PI can enter the apoptotic cells and necrotic cells and effectively stain the concentrated chromatin in apoptotic cells. In addition, for low PI-stained cells, H0342 dye can be used for detection of apoptotic cells with concentrated chromatin. H0342 stain enters cells with intact membranes and stains apoptotic cells more strongly than normal cells. When used in combination with forward scatter (FSC) and side scatter (SSC), H0342 staining

Taken together, the FCM method requires less sample, provides high sensitivity, and simultaneously analyzes apoptotic cells and normal cells. Morphological observation can be carried out using an optical microscope, and electron microscopy is an authoritative method, which is commonly used before serious quantitative analysis. Agarose gel electrophoresis can also be used as a qualitative analysis, but the results need further confirmation by TUNNEL technology or annexin-V/PI

Cell growth (proliferation) can be evaluated by the time-dependent changes

of the total number of proliferating cells, as well as the ratio of cells among individual phases of the cell cycle. The cell growth curve was plotted to show the time-dependent increase in cell numbers. Depending on the cell type and growth condition, the required cell density to enter the exponential phase and the rate of cell growth measured by the doubling time (Tpot) could be very different. For example, most microbials (e.g., *E. coli*) enter the exponential growth phase with low cell numbers and show fast growth rate with a doubling time less than an hour [45],

Additional methods are detecting the release cytochrome C from mitochondria into cytoplasm during apoptosis [42], as well as activation of signaling pathways involving caspase 3, caspase 9, Apaf-1, PARP, Bcl2, Akt, TFAR19, etc., to differenti-

partially necrotic, and the cellular debris could cause false detection.

ate the exogenous or endogenous cause of cell apoptosis [43].

PI dye does not pass through the intact cell membrane, but in the middle and late stages of apoptosis, PI can pass through the cell membrane to bind to the DNA and redden the nucleus [40, 41]. PI staining of the apoptotic cells shows a subdiploid peak (Ap peak) that appears before the G1 peak. The number of apoptotic cells can be detected based on the level of the Ap peak. Therefore, combining results from both annexin V staining and PI staining, cells in the early and late stage of apoptosis

*Cell Growth*

**42**

apoptotic cells.

*evaluate the apoptosis.*

**Figure 3.**

The limitations of the TUNEL method are as follows. Firstly, it labels apoptotic cells only in the middle and late stages. Secondly, necrotic cells also contain DNA breaks and are labeled by dUTP, so the assay is not specific enough to distinguish between apoptotic and necrotic cells. Thirdly, cells need to be fixed during the labeling process, which may lead to excessive cell debris or loss of DNA fragments. Lastly, subjective factors are involved when counting the number of the

*Methods in measuring apoptosis: (A) lymphocytes stained with EB/AO solution. Triangle, arrow, and arrowhead show viable, early apoptotic and late apoptotic cells, respectively. (B) Transmission electron image of a broken apoptotic lymphocyte. The cell breaks up into apoptotic bodies containing organelles or the condensed nuclear fragments. (C) Scanning electron image of a macrophage phagocytosing the apoptotic lymphocytes (asterisks). (D) A macrophage engulfing an apoptotic lymphocyte L, containing the condensed nuclear fragments. Arrows show phagosomes containing the apoptotic bodies of lymphocytes. (E, F) A macrophage phagocytosing apoptotic cells. (G–J) LC3 fluorescent stain in macrophages phagocytosing the apoptotic lymphocytes. The nuclei were stained with DAPI (G) or PI (H). (K and L) PI staining was used to* 

In addition, agarose gel electrophoresis can be used to detect the apoptotic DNA fragmentation into an integer multiple of 180–200 bp [36]. ELISA can be performed using anti-DNA and anti-histone monoclonal antibody to detect nucleosome fragments [37]. Mitochondrial membrane potential measurement can distinguish early

Application of annexin V is a widely used method for detecting apoptosis [39]. Annexin V is a Ca2 + −dependent phospholipid binding protein with a molecular weight of 35–36KD, which can bind with phosphatidylserine with high

apoptosis [38] and provides a nice complement to the TUNNEL method.

**3.3 Fluorescent staining for flow cytometry analysis**

affinity. Annexin V can be labeled with fluorescein isothiocyanate (FITC, PE) or biotin and used as a fluorescent probe to detect apoptosis by flow cytometry or fluorescence microscopy.

PI dye does not pass through the intact cell membrane, but in the middle and late stages of apoptosis, PI can pass through the cell membrane to bind to the DNA and redden the nucleus [40, 41]. PI staining of the apoptotic cells shows a subdiploid peak (Ap peak) that appears before the G1 peak. The number of apoptotic cells can be detected based on the level of the Ap peak. Therefore, combining results from both annexin V staining and PI staining, cells in the early and late stage of apoptosis and necrosis can be distinguished.

However, the limitation of both staining methods is the poor sensitivity. It is difficult to detect DNA fragments in early apoptotic cells and easy to miss the detection of apoptotic cells in S phase or G2/M phase. Meanwhile, the PI method is partially necrotic, and the cellular debris could cause false detection.

Additional methods are detecting the release cytochrome C from mitochondria into cytoplasm during apoptosis [42], as well as activation of signaling pathways involving caspase 3, caspase 9, Apaf-1, PARP, Bcl2, Akt, TFAR19, etc., to differentiate the exogenous or endogenous cause of cell apoptosis [43].

### **3.4 Double or multiple staining**

To distinguish the apoptotic cells, necrotic cells, and living cells, researchers perform flow cytometric analysis after double staining with annexin V and PI. Annexin V-FITC has poor membrane permeability and can specifically bind phosphatidylserine; for live cells, no phosphatidylserine can be detected by annexin V, but during apoptosis, phosphatidylserine is valgus outward to the outer side of the cell membrane for easy detection. The fluorescent dye PI binds to chromatin but does not enter the cytoplasm of live cells. However, PI can enter the apoptotic cells and necrotic cells and effectively stain the concentrated chromatin in apoptotic cells. In addition, for low PI-stained cells, H0342 dye can be used for detection of apoptotic cells with concentrated chromatin. H0342 stain enters cells with intact membranes and stains apoptotic cells more strongly than normal cells. When used in combination with forward scatter (FSC) and side scatter (SSC), H0342 staining can distinguish apoptosis from living cells [44].

Taken together, the FCM method requires less sample, provides high sensitivity, and simultaneously analyzes apoptotic cells and normal cells. Morphological observation can be carried out using an optical microscope, and electron microscopy is an authoritative method, which is commonly used before serious quantitative analysis. Agarose gel electrophoresis can also be used as a qualitative analysis, but the results need further confirmation by TUNNEL technology or annexin-V/PI dual-label method.
