HEK cell DNA content analysis by flow cytometry
Cells (1 x 106) were harvested 72 HPT and fixed in 70% (v/v) EtOH at 4°C for 30 min, with gentle vortexing to prevent clumping. EtOH was removed and the cells washed twice in 1x PBS. Fixed cells were treated with 100 µl RNAse A (100 µg/mL in PBS; Qiagen) for 5 min at RT and the DNA stained with the subsequent addition of 400 µl propidium iodide (PI) (50 µg/mL in PBS; Thermo Scientific) at room temperature for a minimum of 30 min. PI-stained cells were analysed by flow cytometry using an LSRII instrument (BD Biosciences). Gated single cell populations were detected based on PI signal and the resulting histograms analysed using FlowJo software to determine the relative distribution of cells within the cell cycle (G1/S/G2-M) phases.