Cell Cycle Analysis by Flow Cytometry

Cells were treated in three biological repeats for 72 h, collected using 0.25% trypsin in EDTA and washed twice in PBS. Cells were fixed in ice-cold 70% ethanol, washed in ice-cold PBS, and stained in 500 µL PI/Triton X-100 solution, containing 100 µg/mL DNAase-free RNAse A (Sigma Chemical Co.), 50 µg/mL propidium iodide (Sigma Chemical Co.), and 0.25% (v/v) Triton X-100 (Sigma Chemical Co.) in PBS, for 1.5 h at room temperature in dark. Cells are then washed in cold PBS and analyzed by a flow cytometer (BD Accuri C6, BD Biosciences, Franklin Lakes, NJ). Data from at least 10,000 events per sample were collected and presented as proportions of the cells in G0/G1, S, and G2/M phases using ModFit LT 5.0 software (Verity Software House, Topsham, ME). Experiments were repeated in three triplicates.