Opencomet

Comet assay was performed with using Reagent Kit for Higher Throughput Single Cell Gel Electrophoresis Assay (4252-040-K, Trevigen; Gaithersburg, MD, USA).51 (link), 52 (link), 53 (link), 54 (link), 55 (link) The assay was performed according to the manufacturer's instructions, with the following modification: For each sample, 10 μl of cells in a v-shaped, 96-well plate were resuspended with 100 μl low-melting agarose; 15 μl of the cell-agarose suspension was then applied to an appropriate well of the 96-well glass plate. Electrophoresis was performed in a standard, horizontal electrophoresis chamber (26 × 18.5 × 9.5 cm; 550 ml) using 200 mM sodium hydroxide and 1 mM EDTA as the alkaline unwinding buffer. Electrophoresis was run using 380 ml of buffer at 15 V for 45 min at 4 °C. Comets were visualized by staining with SYBRGreen 1:10,000 (S7563, ThermoFisherScientific, Waltham, MA, USA). The plates were washed twice after staining. Images from each well were acquired with an Olympus IX81 Widefield microscope using Olympus ScanR acquisition and then length of the comets was quantified with OpenComet (ImageJ). Mean-TailDNA% was used as a readout. Plates were washed twice after staining. Pictures were taken with Olympus IX81 Widefield microscope using Olympus ScanR acquisition and analysed with OpenComet (ImageJ). Mean-TailDNA% was used as a readout.