4.11. Comet Assay or Single Cell Gel Electrophoresis

Four days after transfection, cells were trypsinized, counted and diluted to give approximately 5 × 10⁴ cells/mL. Eighty microliters of the cell suspension were added to 400 µL of 0.5% low melting agarose maintained at 37 °C, and 90 µL of this suspension were pipetted onto a slide, which was precoated with 1.0% agarose. An additional 1% low melting agarose layer without cells was added after solidification of the layer. Alkaline lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 1% Triton X-100, 10% DMSO, pH to 10.0) was added to the slides at 4 °C for 1 h in the dark to lyse cells. After lysis, the slides were placed in neutralizing solution (400 mM Tris-HCl, pH 7.5) and rinsed three times for 30 min to remove salts and detergents. Slides were then placed in a horizontal electrophoresis chamber with alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13) and incubated for 20–60 min to allow unwinding of the DNA. Electrophoresis of the slides was run for 30 min at 0.6 V/cm. After electrophoresis, the slides were rinsed with neutralization buffer twice and stained with 100 µL ethidium bromide solution (10 mg/mL). Slides were scored immediately or alternatively dried in cold 100% ethanol before storage. DNA tailing due to fragmentation was visualized as comets using an inverted fluorescence microscope and evaluated using TriTek CometScore™ software (TriTek Corp, Sumerduck, VA, USA).