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.