Cometslide assay kits

Neutral comet assays were performed using CometSlide assay kits (Trevigen). Cells were treated with either vehicle (DMSO), 4 mM HU, 50 μM Mirin, or HU + Mirin for 5 h. Cells were embedded in agarose, lysed, and subjected to neutral electrophoresis. Before image analysis, cells were stained with ethidium bromide and visualized under a fluorescence Leica DM5000B microscope using 20× objective lens (numerical aperture of 0.5) with a Leica DFC350FX digital camera and the Leica Application Suite (version 4.1.0). Single-cell electrophoresis results in a comet-shaped distribution of DNA. The comet head contains high molecular weight and intact DNA, and the tail contains the leading ends of migrating fragments. Olive comet moment was calculated by multiplying the percentage of DNA in the tail by the displacement between the means of the head and tail distributions, as described (71 (link)). We utilized the program CometScore, version 1.5 (TriTek) to calculate Olive Comet Moment. A total of 60 to 100 comets were analyzed per sample in each experiment.

Neutral comet assays were performed using CometSlide assay kits (Trevigen). Cells were treated with either vehicle or DEXA for 2 h. The cells were then irradiated with 5 Gy and incubated at 37 °C for different periods of time (0, 30, 60, 90 and 120 min) to allow DNA damage repair. The cells were embedded in agarose, lysed, and subjected to neutral single cell gel electrophoresis. The agarose was dehydrated and the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) for visualization under a fluorescence microscope. The olive comet moment was determined using the software CometScore Version 1.5 (TriTek). The olive moment was calculated by multiplying the percentage of DNA in the tail and the difference between the means of the head and tail distributions, as described [21] (link). A total of 50 comets (and two replications) were analyzed per sample for each experiment. These experiments were performed in triplicate.