Alkaline comet assay (single cell electrophoresis)

Cells were collected with trypsin and washed once in PBS. Cells were then suspended in PBS at approximately 1 x 10⁶ cells/mL and were combined in a 1:30 ratio with CometAssay LMAgarose (R&D Systems 4250-050-02) at 37°C. 60 μL of cell suspension were spread in an even layer on CometSlides (R&D Systems 4250-200-03) and allowed to polymerize at 4°C in a humidified chamber for 30 minutes. Slides were then immersed in pre-chilled lysis buffer (R&D Systems 4250-050-01) overnight. DNA unwinding was performed at room temperature for 1 hour in 300 mM NaOH/3 mM EDTA solution. Following unwinding, electrophoresis was performed at 4°C in prechilled 300 mM NaOH/3 mM EDTA using the CometAssay Electrophoresis System II (R&D Systems, 4250-050-ES) for 30 minutes at 18V/330mA. Slides were then washed 2x in PBS for 5 minutes each. Slides were fixed in -20°C 70% ethanol for 30 minutes. Slides were then washed 2x in PBS for 10 minutes each and 2x briefly in MilliQ water, after which they were then dried at 37°C. DNA was stained using 1:5000 SYBR Gold (Invitrogen S11494) for 30 minutes in the dark. Slides were then washed 2x in PBS for 10 minutes and 2x briefly with MilliQ water before drying again. Coverslips were affixed to slides using clear nail polish before imaging with a Nikon Ti2-E inverted microscope using a 20x/NA 0.75 air objective. Images were processed and tail moments— calculated as (tail length) x (% DNA in the tail— were calculated using the automated OpenComet ImageJ plugin.⁷⁷ Mis-segmented comets were manually removed from the analysis.