Comet Assay Kits, 96-Well

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2x10^5 cells/ml	Protocol for Neutral Comet Assay	Select fifty random cells and analyze the tail moment using OpenComet (open-source automated software)

Upstream tips
Seed 2x10^5 cells/ml
Protocol tips
Protocol for Neutral Comet Assay
Downstream tips
Select fifty random cells and analyze the tail moment using OpenComet (open-source automated software)

Publication protocol

Cells were exposed to 30% H2O2 for five minutes and used as positive controls. In all the experimental samples, the cell viability in Trypan blue exclusion test was acceptable (over 75%). Next, the neutral version of the CometAssay® was performed per the manufacturer’s instructions (Appendix A, Trevigen, Gaithersburg, MD, USA). Without alkaline treatment, this version of the neutral comet assay mainly detects DBS. Additionally, in order to reduce known causes of assay variability, all experiments were run using the Trevigen’s CometAssay® Electrophoresis System. Next, to assess whether DNA damage was related to adrenergic receptor activation, cells were pre-treated for 30 min with propranolol (10 µM) (non-specific β-receptor antagonist) before hormone treatment. Importantly, for every experiment, cell viability was measured using trypan blue exclusion to ensure 75% or more viability per sample. Lastly, DNA was stained with SYBR Green (Trevigen, Gaithersburg, MD, USA), and slides were imaged using Eclipse Ts2r inverted microscope (Nikon Instruments, Mellvile, NY, USA) at 20× magnification. Fifty random cells were selected per sample and analyzed for tail moment using OpenComet (open-source automated software).

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