Dmi5 microscope

hPSCs were subjected to neutral comet assays and performed according to the manufacturer’s protocol (Trevigen, #4260–096). Cells were dissociated with Accutase for 10 min at 37°C. To ensure single-cell dissociation, the cell suspension was gently pipetted up and down washed four times with PBS. Cells were then pelleted, resuspended, filtered through 15-μm cell strainer and seeded onto the 96-well Comet chip (5,000 cells/well) containing E8 Medium supplemented with DMSO, Y-27632, CloneR (STEMCELL Technologies), RevitaCell (Thermo Fisher Scientific), SMC4 (BioVision) or CEPT. Cells were allowed to settle and were incubated for 6 h. Subsequently, the 96-well comet chip was rinsed, overlayed with LMAgarose (Trevigen) and allowed to set for 3 min. Comet chip was placed in a lysis solution for 2 h at 4°C, equilibrated in neutral solution and subjected to electrophoresis at 4°C for 50 min at 22 V in neutral solution. Comet chip was stained overnight at 4°C in 0.2X SYBR Gold (Thermo Fisher Scientific). Comets were visualized using a Leica DMi5 microscope using the appropriate filters. Comets were analyzed using the Comet Analysis Software 1.3d (Trevigen).