Quantifying DNA Double-Strand Breaks via Neutral Comet Assay

To assess the amount of DNA DSBs generated by siRNA treatments, a neutral comet assay was used. Cells were cultured in a six-well plate and treated as outlined above. For the comet assay, these cells were then incubated with 0.4 mL of Trypsin for 10 min. One milliliter of medium was added so that the cells could be suspended and then centrifuged at 1000 × g for 5 min. The cells were then resuspended in 500 μL of fresh PBS and added to prewarmed low-melting point agarose at 37 °C (10 μL of cells to 90 μL of agarose). This suspension was pipetted onto a CometSlide (4250, Trevigen) and spread equally before being allowed to set for 30 min at 4 °C. The slides were then submerged in cold Lysis Buffer (4250, Trevigen) for 30 min on a shaker and then in cold Tris/Borate/EDTA buffer. Electrophoresis was run for 15 min at 21 V in a Mini-Sub Cell GT electrophoresis tray (Bio-Rad). Subsequently, cells were fixed in 70% ethanol and allowed to dry overnight. DNA was stained with Cygreen (GEN-105, ENZO) for 30 min, and slides were imaged on an EVOS fluorescence microscope (AMG) with appropriate filters for green fluorescent protein imaging. Images acquired were processed using the Open Comet software and the olive moment for each group of cells was calculated^{98 (link)}.