Sybr green 1 dye

The comet assay (Trevigen, Gaithersburg, MD) was performed as described previously [44 (link)]. Briefly, T98G cells were treated, trypsinized and washed with ice-cold PBS. Next, the cells at 1 × 10⁵/ml were embedded in LMAgarose and immediately pipetted 100 μL onto CometSlide. After cooling, submerse the slides flat in pre-cooled lysis buffer and freshly prepared Alkaline Unwinding Solution, pH > 13. Electrophoresis was carried out at 25 V, 300 mA. The slides were washed in neutralization buffer (0.4 M Tris-HCl, pH 7.5) for three times and in 70% ethanol. Subsequently, the DNA was stained with SYBR Green I dye (Sangon Biotech, 1:10,000 in Tris-EDTA buffer, pH 7.5) and images were captured using a fluorescence microscope (Nikon, Japan).

The comet assay (Trevigen, Gaithersburg, MD) was performed according to the manufacturer’s protocol using alkaline conditions. Cell samples were handled under dimmed light to prevent DNA damage from ultraviolet light. Combine cells at 1 × 10⁵/ml with molten LMAgarose and immediately pipette 50 μl onto CometSlide. After placing slides flat at 4 °C for 10 min, immerse slides in lysis solution for 60 min and freshly prepared Alkaline Unwinding Solution, pH > 13 for 20 min. Electrophoresis was carried out at the rate of 1.0 V/cm for 30 min. The slides were removed from the electrophoresis chamber, washed in deionized water for 5 min and in ice cold 70% ethanol for 5 min. Subsequently, the slides were air-dried, and DNA was stained with 50 μl of SYBR Green I dye (Sangon Biotech, 1:10,000 in Tris-EDTA buffer, pH 7.5) for 30 min and immediately analyzed using a fluorescence microscope (Axiovert 200, Carl Zeiss). Data was analyzed using CometScore (TriTek, Sumerduck, VA).