2.19. Cell Cycle Analysis

PyMT EV or sh61 KD cells were plated into 6-well dishes, and when sub-confluent, they were treated with vehicle control or with 25 mM cCr for either 24 h (added 24 h prior to experiment endpoint) or 48 h. Harvested cell pellets were washed twice with cold PBS and re-suspended in 5 mL ice-cold 70% ethanol prior to storage at 4 °C. Prior to cytometry, samples were washed with PBS and treated with RNaseI. Setting aside an unstained sample as the gating control, propidium iodide [50 μg/mL] was added and incubated at 37 °C for 15 min prior to analysis on a BioRad ZE5 flow cytometer at the UTHSC Flow Cytometry and Cell Sorting (FCCS) Core. Raw data were exported to ModFit for analysis (n = 3 replicates/genotype/time point, and at least duplicate biological replicates per time point).