Alkaline Comet Assay for DNA Damage

Alkaline comet assay was performed as described previously by [74 (link)] with some modifications. Briefly, cells were seeded in tissue culture flasks and incubated for 12 and 24 h with 3a at 10 μM. After checking the cell viability rate (at least 70% of viable cells), two hundred thousand cells were suspended in 0.5% low melting point agarose (Sigma-Aldrich). Cellular suspension was spread into microscope slides pre-coated with 1.5% normal-melting point agarose (Sigma-Aldrich). After lysis, electrophoresis was performed. Finally, the samples were neutralized, fixed, and stained with SYBR^® Green I solution (Invitrogen by Thermo Fisher Scientific Inc., Rockford, IL, USA). The analysis was performed using a fluorescence microscope at 20× magnification. The images were analyzed using the ImageJ [73 (link)] plugin OpenComet [75 (link)] to demarcate the “head” and the “tail” regions of each comet. Samples treated with ultraviolet light were used as a positive control. Fifty randomly selected nuclei were analyzed sample. The analysis was done by the extent of DNA damage (tail moment).