**3. Apoptosis**

*Cell Growth*

beled 3

**2.2 DNA content analysis**

**2.3 Protein biomarkers**

4′,6-diamidino-2 phenylindole (DAPI)

Propylene glycol monomethyl ether acetate (PGMEA)

Ethidium bromide monoazide (EMA)

3 HTdR incorporation <sup>3</sup>

*Comparison of methods for DNA labeling.*

[30], and phosphorylated histone H3 [31].

incorporation as shown in **Table 1**.

DNA content can be analyzed after fluorescent staining or labeled nucleic acid

As shown in **Figure 2**, DAPI and Hoechst dyes penetrate the membrane and are commonly used to label live cells, whereas propidium iodide (PI), propidium monoazide (PMA), and ethidium bromide monoazide (EMA) only label dead cells. Newly synthesized DNA in active proliferating cells can be labeled by the radiola-

HTdR, 125IUdR [24], or the fluorescent-labeled BrdU and EDU [25]. Flow cytometry measurement (FCM) is a sensitive method to measure cell size and fluorescent labeling. Double staining both cellular DNA and protein biomarker allows identification of cells in G1 peaks, G2+ M peaks, and S platforms, as well as the subdiploid peaks (apoptotic peaks) before the G1 peak. These methods in combination with other biomarkers show the distribution of cells in each phase of

Proteins that are found in proliferating cells, but not in nonproliferating cells can be used as biomarkers for cell cycle measurement. Ki-67 protein (also known as MKI67) is present during all active phases of the cell cycle (G1, S, G2, and mitosis) but is absent in resting (quiescent) cells (G0) [27]. During interphase, the Ki-67 protein is exclusively located in the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. During cell progression through S phase of the cell cycle, the Ki-67 protein markedly increases [28]. As shown in **Figure 1**, the fluorescent-labeled monoclonal Ki-67 antibody has been used for cell cycle measurement and cancer diagnosis. Other commonly used cell proliferation biomarkers include proliferating cell nuclear antigen PCNA [29], topoisomerase IIB

**DNA stain Principle Membrane** 

Hoechst dye Binds to DNA A-T rich region [21] Yes Live cell Propidium Iodide (PI) Label dead cells [22] No Dead cell

> In combination with dsDNA to form a stable and strong covalent nitrogen-

> Covalent cross-linking with genomic

125IUdR incorporation 125IUdR incorporates in DNA synthesis Yes Live cell

carbon bond [23]

DNA [23]

BrdU incorporation Brdu participates in DNA synthesis in cell proliferation

EDU incorporation EDU participates in DNA synthesis in cell proliferation

**permeability**

No Dead cell

No Dead cell

Yes Live cell

Yes Live cell

Binds to DNA A-T rich region [20] Yes Live cell

H-TdR incorporates in DNA synthesis Yes Live cell

**Function**

the cell cycle and can be used to investigate cell dynamics [26].

**40**

**Table 1.**

Apoptosis is a programmed cell death, and the process involves a series of morphological changes such as blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, as well as biochemical changes such as chromosomal DNA fragmentation, global mRNA decay [32], and appearance of protein biomarkers in protein degradation pathways.

Commonly used analyses include transmission electron microscopy for morphological analysis, biochemical assays for detection of apoptotic biomarkers, and flow cytometry analysis of cellular DNA content (**Figure 3**).

### **3.1 Transmission electron microscopic analysis**

Tissue or cells can be directly stained using dyes such as hematoxylin, methyl green pyronine, and acridine orange for microscopic analysis. Under the transmission electron microscope [33], apoptotic cells show reduced size and more concentrated cytoplasm. In the nucleus of pro-apoptosis phase, the chromatin is highly coiled, and many vacuole structures called cavitation appear; in the phase IIa nucleus, the chromatin is highly coagulated and marginalized; in the end, the nucleus is cleaved into fragments and produces apoptotic bodies. Fluorescence microscopy and confocal laser scanning microscopy [34] can also be used to observe the progress of apoptosis, based on morphological changes of nuclear chromatin DNA-specific dyes such as Hoechst dye series and DAPI.
