Reagent kit for higher throughput single cell gel electrophoresis assay

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.

Comet assay was performed with the Reagent Kit for Higher Throughput Single Cell Gel Electrophoresis Assay (4252-040-K, Trevigen; Gaithersburg, MD, USA). The assay was performed according to the protocol, but with the following changes. For each sample 10 µL of cells were resuspended with 100 µL LMAgarose, and 15 µL were spread on the comet plate. To adjust for the used electrophoresis apparatus, the alternative alkaline unwinding and electrophoresis solutions were used. Electrophoresis was run with 380 mL at 15 V for 1 hour in an ice bath. Comets were stained with SYBRGreen 1:10 000 (S7563, ThermoFisherScientific, Waltham, MA, USA). Plates were washed twice after staining. Pictures were taken with Olympus IX81 Widefield microscope using Olympus ScanR acquisition, and comets were analyzed with OpenComet (ImageJ). Mean TailDNA% was used as readout.