Neutral Comet Assay for DNA Double-Strand Breaks

A neutral comet assay was used to specifically assess the amount of DNA DSBs in cells treated with Veliparib, CPT, or both. In individual wells of a six-well plate, cells were incubated with 0.4 mL of Trypsin for 10 minutes. 1 mL of medium was added to the trypsin and the cells centrifuged at 1000 g for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 500 μL of PBS. The suspended cells were added to prewarmed low-melting agarose at 37°C (10 μL of cells to 90 μL of agarose). The mix was pipetted onto a CometSlide (4250, Trevigen) and spread equally across the slide before being allowed to set for 30 minutes at 4°C. Following this, the slides were submerged in cold Lysis Buffer (4250, Trevigen) for 30 minutes on a shaker, and then in cold Tris/Borate/EDTA buffer. Electrophoresis was run for 15 minutes at 21V 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 minutes, and slides were imaged on an EVOS fluorescence microscope (AMG) with appropriate filters for GFP imaging. Images acquired were processed using Open Comet software and the olive moment for each group of cells was calculated [83 (link)].