Comet Assay for DNA Damage

After exposure, the cells were digested, resuspended in a culture medium, and then placed on ice. The sandwich agarose gels were made up of 0.65% normal-melting agarose that had been pre-coated on a slice and 0.65% low-melting agarose mixed with cells on normal-melting agarose. The cells were lysed in lysis buffer containing 1% Triton X-100 at 4°C for 1 h and then enzymatically hydrolyzed in lysis buffer containing 0.5 mg/ml DNase-free proteinase K (Beyotime, Shanghai, China) at 37°C for 2 h. Before electrophoresis, DNA in cells was unwind in ice-cold alkaline electrophoresis solution for 20 min and then electrophoresis at 20 V for 20 min. After electrophoresis, cells were neutralized in Tris buffer (0.4 M, pH 7.5) for 2 × 5 min. DNA “comets” were stained by Gel-red (Beyotime) and photographed by a fluorescence microscope (Zeiss, Oberkochen, German). Approximately 30 comets for each sample were calculated using the CASP 1.2.2 software (Krzysztof Konca, Wroclaw, Poland). Additional details can be found in a previously described protocol (32 (link)).