Annexin V/propidium iodide (PI) staining and flow cytometry

The experiment was performed according to the instruction manual of the Annexin V-FITC Apoptosis Detection kit (Beyotime Institute of Biotechnology). Briefly, adherent cells were digested using trypsin. The cells were washed with PBS once, centrifuged, and gently resuspended in 195 µl of Annexin V-FITC binding solution. Next, 5 µl of Annexin V-FITC was added, and the sample was gently mixed. Finally, 10 µl of PI staining solution was added, and the sample was gently mixed and incubated at 20°C in the dark for 30 min. The cells were then detected using a flow cytometer (Cytomics FC 500; Beckman Coulter, Inc.) and analysed using FlowJo software (Version 7.6.1; FlowJo, LLC).