Flow cytometry analysis of apoptosis and the cell cycle

Cells were collected and washed twice with cold PBS, and they were then prepared according to the protocol of the Annexin V-FITC Apoptosis Detection Kit. In cell cycle assays, the cells were first synchronized by serum starvation for 24 h. After drug treatment (48 h), the cells were washed with ice-cold PBS and dispersed before adding pre-cooled 70% ethanol (stored at −20 °C overnight). Then, cells were washed with PBS and incubated with 500 μL PI/RNase solution for 15 min. The prepared cellular samples were immediately analysed on a Coulter Epics XL Flow Cytometric System and processed by Summit 5.2 (Beckman Coulter, Miami, FL, USA).