Cell apoptosis assay

An Annexin V-FITC/PI Apoptosis Assay Kit (Invitrogen Company, United States) was used to assess cell apoptosis. The cells were digested with trypsin, followed by washing twice with PBS. The 1 × 10⁶ cells /mL cells were centrifuged for 5 min, and the supernatant discarded. Annexin-V-FITC labeling solution (20 μL) and PI reagent (20 μL) were successively added into 1 mL of buffer containing the cells, and incubated at room temperature in the dark for 5 min. A Beckman Coulter CytoFLEX LX Flow Cytometry System was employed to measure cell apoptosis. The experiment was repeated three times, and the data were averaged.