**5.1 DNA ladder assay**

*Cell Growth*

concentration.

**5. Detection of apoptosis by comet assay**

removed. Cells will be treated with varying concentration of compounds diluted with medium. The plates can be incubated for 72 h. After incubation, the media will be removed without disturbing the cells, and to each well, 100 μl of 5 mg/ml stock solution of MTT will be added, and the plates can be incubated for 2 h in the dark at 37°C in a CO2 incubator. About 100 μl of lysis buffer will be added to each well, and the plates can be further incubated for 4 h in the dark in a CO2 incubator, and absorbance can be read using ELISA plate. Three replicates will set up for each

Comet assay can be done for the quantitation of low levels of DNA damage in individual [22]. Cancer cells except the control cells can be treated with various drugs. Then it can be centrifuged to get the pellets, and pour 1% NMPA agar on the base slides using filler pipette and allow to solidify. Then the slides were kept in a polar ice pack. The pellets thus obtained will be taken and is mixed with 200 μl of 0.5% LMPA agar. About 15 0 μl cells from the above tubes were taken and poured over the base slides, and allow to solidify after placing a cover slip over it, and then remove the cover slip and pour a layer of 1% LMPA agar over it, and allow to solidify and keep these slides in a coupling jar containing lysing solution. It will be kept in refrigerator for 1:30 h, and subject it to electrophoresis for 20–30 min in electrophoresis apparatus at 25 V. Then the slides were taken and washed with neutralizing buffer solution for three times. Pour ethidium bromide stain over the slides, and view through fluorescent microscope. The figure shows the comet assay on KB human oral cancer cells after treatment with compound for 24 h (**Figure 3**).

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**Figure 3.**

*Comet assay: (a) non-apoptotic cells, (b) comet-shaped apoptotic cells.*

Breakdown of genomic DNA into multiples of approximately 180 bp is considered to be a hallmark of apoptosis [14]. This cleavage of chromosomes produces a large number of DNA breaks, and subsequently a simultaneous amount of new 3′-OH DNA ends. In normal living cells, only an insignificant number of 3′-ends are present; this helps to detect apoptosis. The enzyme terminal deoxynucleotidyl transferase (TdT) has the capability to incorporate individual deoxyribonucleotide triphosphates to the 3′-end of double- or single-stranded DNA. This quality can be detected using 3′-ends with nucleotides that have been labeled with radioactive, fluorescent, or digoxigenin labels. Apoptosis can be measured quantitatively by using gel electrophoresis; here the apoptotic DNA is organized into a typical ladder pattern of multiples of 180 bp. In situ labeling of 3′-ends can be used to detect qualitatively apoptotic cells (**Figure 4**).
