Trevigen neutral comet assay kit

Cells were irradiated at a dose of 400 rads (4Gy) and cultured in complete media for desired duration. At desired time points after irradiation, cells were harvested and processed using the Trevigen neutral COMET assay kit following the kit protocol (Trevigen, MD, cat. # 4250–050-K). Cells were mixed with low melting point agarose and pipetted onto pre-coated slides and placed at 4°C until solidified. Then slides were placed in pre-chilled lysis solution overnight at 4°C. Following lysis, slides underwent gel electrophoresis in 1x Neutral Electrophoresis Buffer at 17 V for 45 min. The slides were then immersed in DNA Precipitation Solution for 30 min at RT, followed by immersion in 70% Ethanol for 30 minutes at RT. Dried slides were then stained with SYBR Green (Fisher, cat. # S7563), rinsed briefly in water, and dried at 37°C. Slides were visualized with the EVOS Cell Imaging System (ThermoFisher Scientific). Images were taken with the EVOS 10x objective and quantified for comets flagged by the OpenComet software as “normal”. Some abnormal comets were manually excluded.

Neutral comet assays were performed using the Trevigen Neutral Comet Assay Kit according to the manufacturer’s instructions (Trevigen). Briefly, cells were mixed with melted LM Agarose (Trevigen). Neutral electrophoresis was conducted with 1X Neutral Electrophoresis Buffer at 21 V for 1 h at 4°C in the Comet Assay Electrophoresis System (Trevigen). The DNA was stained with SYBR Green I (Trevigen) and observed by fluorescence microscopy. Comet images were analyzed using CASP software (CASP, Wroclaw, Poland) [25 (link)]. Data are presented as the mean±SEM for 3 biological replicates with more than 40 cells analyzed per replicate.