Comet Assay

Comet Assay

Neutral COMET assays (Jami, 2014)

The neutral COMET assays were performed in accordance with manufacturer’s (Trevigen) instructions. Briefly, cells were seeded the day before treatment at 1x105cells per well in a 6-well tissue culture dish. On the day of treatment, cells were treated with 1 mM camptothecin, 3 mM HU, and 5 mM ATRi as necessary and harvested by trypsinization. Cells were washed once with cold PBS then resuspended at 2x105 per mL in cold PBS. During this time, low melting temperature agarose (Trevigen) was melted and held in a 42 degree water bath. To prepare slides, 5 mL of cell suspension was mixed with 50 mL of agarose and spread into one well of a COMET slide (Trevigen). Two wells were prepared per treatment condition. Slides were allowed to gel for 15 minutes at 4 degrees. Slides were then immersed in pre-chilled Lysis Buffer (Trevigen) for 1 hour at 4 degrees. Slides were rinsed once with pre-chilled TAE (40 mM Tris Base, 20 mM Acetic acid, 1 mM EDTA, pH 8.45), then washed for 30 minutes immersed in TAE at 4 degrees. Slides were then electrophoresed for 1 hour at 1 V/cm immersed to a depth of at least 0.5 cm in TAE. After electrophoresis, slides were immersed in DNA Precipitation Solution (1M NH4Ac, 87% EtOH) for 30 minutes at room temperature. Next, slides were immersed in 70% ethanol for 30 minutes at room temperature then dried for 15 minutes at 45 degrees and stored overnight at room temperature. Slides were stained with 100 mL of 1X SYBR Green I (Trevigen) for 30 minutes at room temperature. SYBR Green solution was decanted and slides allowed to dry at least 30 minutes before visualizing slides using a Zeiss Axioplan microscope. 20 images were taken per well and scored for tail moment using CometScore software (http://www.autocomet.com). At least 100 cells were scored for each condition.