CometAssay Single Cell Gel Electrophoresis Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 1,00,000cells/ml	For neutral comet assay, the slides are to be immersed in 1 × TBE buffer for electrophoresis at 1 volt/cm for 30 min at room temperature. For both alkaline and neutral comet assays, the slides can be immersed in 70 % EtOH for 5 min after electrophoresis then incubated with SYBR®Gold for 30 min.	The images can be visualized under BioTek Cytation 5 cell imaging reader. Statistical analysis was performed by OpenComet, an ImageJ plugin

Upstream tips
Seed 1,00,000cells/ml
Protocol tips
For neutral comet assay, the slides are to be immersed in 1 × TBE buffer for electrophoresis at 1 volt/cm for 30 min at room temperature. For both alkaline and neutral comet assays, the slides can be immersed in 70 % EtOH for 5 min after electrophoresis then incubated with SYBR®Gold for 30 min.
Downstream tips
The images can be visualized under BioTek Cytation 5 cell imaging reader. Statistical analysis was performed by OpenComet, an ImageJ plugin

Publication protocol

The comet assays were performed according to the Trevigen CometAssay™kit protocol with slight modifications. Cells were pre-treated with PARGi for 1 h, followed by co-treatment with 20μM CPT for 2 h. Treated cells were trypsinized at 37 °C for 5 min. An equal amount of drug-free medium was then added to quench the trypsin activity. The cells were spun down and resuspended in fresh PBS. The final cell density was approximately 100,000cells/ml. Fifty microliters of the cell suspension were then mixed with 500μl of 0.5%low melting point agarose (Invitrogen) (in PBS) at 37 °C. Fifty microliters of the cell/agarose mixture were transferred onto glass slides. The slides were then immersed in prechilled lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH10.0, 1% Triton X-100, and 10% Me2SO) for 1 h. For the alkaline comet assay, the slides were immersed in an alkaline unwinding solution (200 mM NaOH, 1 EDTA) for 30 min at room temperature, followed by electrophoresis in 4 °Calkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA) at 1 volt/cm for30 min. For neutral comet assay, the slides were immersed in 1 × TBE buffer for electrophoresis at 1 volt/cm for 30 min at room temperature. For both alkaline and neutral comet assays, the slides were immersed in 70 % EtOH for 5 min after electrophoresis then incubated with SYBR®Gold for 30 min. The images were visualized under BioTek Cytation 5 cell imaging reader. Statistical analysis was performed by OpenComet, an ImageJ plugin.

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