Alkaline and Neutral Comet Assay

The comet assay was performed using the Comet Assay Kit (ab238544, Abcam plc., Cambridge, UK) according to the manufacturer’s protocol and previously described method^{21 (link)}. In brief, mouse kidneys were removed and minced in a small amount of ice-cold PBS containing 20 mM EDTA. After removing the large pieces of tissue, the supernatant was passed through a 35-μm cell strainer. After centrifugation, the pellet was suspended at 1 × 10⁵ cells/ml in ice-cold PBS. Cell samples were mixed with comet agarose in a 1/10 ratio (v/v) and immediately transferred onto the slide glasses covered with comet agarose base layer. After incubating with pre-chilled lysis buffer, the slides were subjected to electrophoresis. Electrophoresis was performed in the Alkaline Electrophoresis Solution for the alkaline comet assay and in the TBE Electrophoresis Solution for the neutral comet assay. After electrophoresis, the slides were incubated with Vista Green DNA dye. Images were obtained by epifluorescence microscopy (IX71; Olympus, Tokyo, Japan) using the FITC filter. Ten pictures (5–15 cells per picture) were randomly taken, and the tail moment (tail length x tail % DNA/100) of more than 50 cells per group was calculated using Comet Score analysis software (TriTek Corp.).