Flow cytometry
The apoptosis ratio was estimated using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 for flow cytometry, following the manufacturer’s instructions (Invitrogen, Carlsbad, USA). Cells were collected, washed twice with cold PBS, and incubated with Annexin V-FITC mixed with propidium iodide for 10 min in the dark. Cellular fluorescence was assessed using flow cytometry (CytoFLEX S, Beckman, China).