Cell cycle analysis by flow cytometry
First, we washed cells with cold PBS, then trypsinized. The cells were then centrifuged at 2000 rpm for 5 min, and the supernatant was carefully aspirated leaving approximately 50 µL of residual solution. Cells were then resuspended in cold PBS and washed twice. The cell pellet was resuspended in 1 mL of pre-cooled ethanol and fixed at 4 °C for at least 2 h or overnight. After fixation, the cells were centrifuged and the supernatant was removed. Fixed cells were washed twice with pre-cooled PBS and resuspended in 0.5 mL of RI/RNase staining buffer (550825, BD Biosciences, USA) for 15 min at room temperature in the dark. Stained cells were then analyzed using a flow cytometer (CytoFLEX, BECKMAN) equipped with appropriate lasers and filters.