Comet assay kit

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.).

Bacterial suspensions of V. parahaemolyticus ATCC 17802 were prepared according to the method of Kim et al. [12 (link)] with a slight modification, using the Comet Assay Kit (Abcam ab238544, Shanghai, China). Here, 75 μL of the aliquot was pipetted on comet slides, then slides were placed at 4 °C for 15 min to fix the agarose. Thereafter, a 1 h immersion of the comet slides was accomplished in pH 10 Lysis Buffer under dark conditions at 4 °C, and then a further 30 min immersion proceeded in alkaline solution for the slides under the same conditions. Electrophoresis was performed for 20 min (12 V, 100 mA) using alkaline electrophoresis buffer. The slides were then subjected sequentially to three washings with distilled water, each for 2 min, a 10 min dehydration using 70% (v/v) chilled ethanol, drying in air, and subsequent staining with 100 μL of Vista Green DNA Dye staining solution for each well. A Leica DM6 B epifluorescent microscope (Wetzlar, Germany) was utilized in conjunction with Vista Green DNA Dye (WB, 450–480 nm) for acquisition of micrographs at a magnification of 1000×.

The comet assay kit (Abcam, Cambridge, MA, USA) was used to measure cellular DNA damage. This assay is a single-cell gel electrophoresis method for the simple evaluation of cellular DNA damage. First, a base layer of comet agarose was created on a slide, followed by a layer of cells, agarose, and lysis. Next, electrophoresis was performed under neutral conditions, and the cells were stained with DNA dye. Finally, cell morphology was observed by fluorescence microscopy (Olympus IX71/DP72).

The Comet Assay Kit (3-well slides; Abcam; ab238544) was used for checking cellular DNA damage. The assay was performed according to the manufacturer's instructions, and cell morphology was observed using a fluorescence microscope (Olympus IX71/DP72).

The alkaline comet assay was conducted to measure the DNA damage level in satellite cells. Sorted satellite cells were treated with UV radiation at different dosages and the comet assay was operated immediately after irradiation through a Comet Assay Kit (Abcam, ab238544) according to the manufacturer’s instructions. The amount of DNA damage was indicated by the tail moment which is a measure of DNA fragments with a Leica fluorescence microscope.