CometAssay Single Cell Gel Electrophoresis Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 1–2 × 10^4 cells/ml mixed in 1% low fusion point agarose and then place on a slide		The length of tail, the percentage of DNA in the tail of each comet can be measured using CASP software (Version 1.2.3; available at http://casp.sourceforge.net)

Upstream tips
Seed 1–2 × 10^4 cells/ml mixed in 1% low fusion point agarose and then place on a slide
Downstream tips
The length of tail, the percentage of DNA in the tail of each comet can be measured using CASP software (Version 1.2.3; available at http://casp.sourceforge.net)

Publication protocol

Single cell gel electrophoresis (Neutral) was performed with Comet Assay kit (Catalog No. 4250-050-K, Trevi-gen, Gaithersburg, MD) according to manufacturer’s instructions. Briefly, A549 cells were irradiated at 6 Gy and were collected at 0, 2 and 24 h after irradiation, fol-lowed by washing and resuspension in pre-cooling 4 °C PBS. 1–2 × 10^4 cells were mixed in 1% low fusion point agarose and placed on a slide. The cells were then treated with lysis solution for 30 min, and rinsed in EDTA unwinding solution, and then subjected to alkaline elec-trophoresis for 30 min (25 V, 300 mA). Then 50 cells per slide were counted and analyzed under fluorescence microscopy (Olympus FV1000). The length of tail, the percentage of DNA in the tail of each comet were meas-ured using CASP software (Version 1.2.3; available at http:// casp. sourc eforge. net).

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