Comet Assay Protocol for DNA Damage Analysis

Neutral comet assays were performed using CometAssay (Trevigen) according to the manufacturer's protocol with minor modifications (Carvajal-Maldonado et al., 2019 (link)). Upon harvesting, cells were suspended at 3×10⁵ cells/ml in cold PBS before combining with LMAgarose (Trevigen, 4250-50-050-02), spread onto a comet slide (Trevigen, 4250-200-03) and allowed to dry. Slides were placed in lysis solution (Trevigen, 4250-050-01) at 4°C for 1 h. Lysed slides were immersed in 1× TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2.5 mM EDTA) for 30 min before electrophoresis at 25 V for 30 min at 4°C. Slides were washed in DNA precipitate solution (1 M ammonium acetate, 95% EtOH) for 30 min, followed by a fixing step in 70% ethanol for 30 min, and dried overnight at RT. Comets were stained with 1× SYBR Gold (Thermo Fisher Scientific) for 30 min. Images were acquired with a fluorescence microscope (Leica DMU 4000B; 63×/1.40-0.60 NA oil) with a Leica DFC345FX camera. At least 150 comets were scored in each experiment using the OpenComet plugin in the ImageJ analysis software.