Single cell gel electrophoresis assay kit

Comet assays were carried out with the Single Cell Gel Electrophoresis Assay-kit (Trevigen). U2OS cells were transfected with control or Rpp29 siRNAs. After 72 h of transfection, cells were exposed to 10 Gys of ionizing radiation from an X-ray machine (Faxitron, CellRad), followed by recovery for 1, 3 and 6 h. Approximately, 3 × 10³ cells were combined with molten LM Agarose at a ratio of 1:10 and then were applied onto comet slide for 30 min at 4 °C in the dark to solidify. Cells were lysed by incubation with lysis solution for 1 h at 4 °C and slides were run for 30 min at 13 volts. Slides were incubated for 30 min at RT with DNA precipitation solution and fixed with 70% ethanol for 30 min at RT. Samples were dried by incubation for 30 min at RT and stained with SYBR-green. Comet images were acquired using Nikon Eclipsed E400 Epi-fluorescence microscope and percentage of DNA in the comet tail was measured using commercially available COMET SCORETM (TriTeK Corporation) software.

The experiment was performed under alkaline conditions as previously described using the Single Cell Gel Electrophoresis Assay kit from Trevigen (cat # 4250-050-K, Gaithersburg, MD, USA). Fluorescent images were taken using the Cellsens software on a 10× objective of an Olympus IX70 microscope and analyzed by ImageJ (ImageJ 1.8.0, Bethesda, MD, USA) under the OpenComet plugin (http://cometbio.org/, accessed on 17 August 2021) [37 (link)]. All images were taken with the same exposure time and magnification. Data were expressed by the tail DNA percentage (tail intensity out of total DNA intensity).

Alkaline and neutral comet assays were performed using a reagent kit of Single-Cell Gel Electrophoresis Assay kit (4250-050-k, Trevigen, Gaithersburg, MD, USA). Image data were analyzed by CometScore software (v1.6, TriTek Corp, Sumerduck, VA, USA) for the measurement of percentage of tail DNA.

Comet assays were performed using Single Cell Gel Electrophoresis Assay kit (4250-050-ES from Trevigen) according to the manufacturer’s instructions. Comets were then imaged using an inverted Eclipse Ti-E Nikon videomicroscope equipped with a 40× CFI Plan Fluor objective. Images were analysed with OpenComet plugin on Fiji. Based on the comet DNA content of DMSO treated cells, a manual threshold was applied to identify diploid from tetraploid cells. The same threshold was applied on the cells treated for mitotic slippage.

Alkaline comet assays were performed on MCF7 TamR cells using a Single Cell Gel Electrophoresis Assay Kit (Trevigen, Inc., USA) according to the manufacturer’s instructions. One hundred cells were spotted onto each sample area, and 50 cells from each group were analyzed and quantified using the CASP1.2.3 beta1 software (Krzysztof Konca, Comet Assay Software Project Lab, http://caspla.com).

Neutral comet assays were conducted with a Single Cell Gel Electrophoresis Assay kit (4250-050-K; Trevigen) according to the manufacturer’s protocols with minor modifications. A total of 5 × 10⁵ cells were resuspended in 0.5 ml of ice-cold PBS. The cell suspension (10 μl) was blended with 100 μl of warmed LMAgarose (4250-050-02; R&D Systems), and the mixture (50 μl) was then swiftly spread on slides as evenly as possible. DNA was stained with SYBR Gold (S-11494; Life Technologies), and comet tail lengths in 25–50 cells per condition were automatically estimated from the images using OpenComet V1.3 software in the image processing program ImageJ.