Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

EdU labeling was done before antibody staining. Cells were centrifuged, supernatant aspirated, and incubated in PBS with 1 mM CuSO4, 100 mM ascorbic acid (fresh), 1 uM Alexa Flour-647-azide (Life Technologies) for 30 minutes, RT in the dark. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/558783 doi: bioRxiv preprint Alexa Flour 647 (1:1,000, Jackson ImmunoResearch, for γH2AX) in B-PBS with 0.5% NP-40 for 1 hour at 37°C in the dark. Then B-PBS with 0.5% NP-40 was added, mixed, and samples centrifuged. Finally, supernatant was aspirated, cells were incubated in 1 ug/mL DAPI (Sigma Aldrich) and 100 ng/mL RNAse A (Sigma Aldrich) in B-PBS with 0.5% NP-40 overnight at 4°C in the dark. For negative control samples used to draw positive/negative gates, cells were not incubated with EdU but were labeled with Alexa Flour-647-azide, and were not incubated with primary antibody but were labeled with secondary antibody and DAPI.