CometAssay Single Cell Gel Electrophoresis Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2 × 10^5 cells/ml		Use TriTek Comet Image program to analyze the fluorescence images to circumscribe the “head” and the “tail” regions of each comet

Upstream tips
Seed 2 × 10^5 cells/ml
Downstream tips
Use TriTek Comet Image program to analyze the fluorescence images to circumscribe the “head” and the “tail” regions of each comet

Publication protocol

The determination of DNA double-strand-breaks was described in previous literature. The assay was performed following the manufacturer’s protocol for the neutral comet assay utilizing a CometAssayTM Kit (Trevigen, Gaithersburg, MD, USA). 13-AC at different concentrations (0, 2.5, 5, and 10 μg/mL) were added to the cancer cells (2 × 10^5 cells/mL) for 3 h. Low melting point agarose (1%) was mixed with the cells were combined at a ratio of 1:10 (v/v). A portion of the mixture (75 μL) was added onto CometSlideTM and left to settle at 4 °C in the dark. For 30–60 min, the slides were immersed in ice-cold lysis solution (Trevigen). A horizontal electrophoresis was used, and the slides were electrophoresed in 1× TBE (90 mM Tris-HCl, 90 mM boric acid, and 2 mM EDTA, pH 8.0) at 20 V for 10 min. To visualize cellular DNA, the samples were fixed in ethanol (70%) and dried before staining with 1:10,000 SYBR Green I (Trevigen). TriTek Comet Image program was utilized to analyze the fluorescence images to circumscribe the “head” and the “tail” regions of each comet. The integrated fluorescence values of each defined area were recorded. From the trailing edge of the nucleus to the leading edge of the tail, the comet length was determined. The comet length was used as an indication of the extent of the DNA damage. Calculations were averaged per replicate.

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Manufacturer protocol

Download the product protocol from Bio-Techne for CometAssay Single Cell Gel Electrophoresis Assay below.

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