Mini sub cell gt electrophoresis tray

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)].

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)}.