Facscan 420

After stimulation, HepG2 cells were collected, a flow-based Annexin V/fluorescein isothiocyanate (FITC) assay (BD) was used to measure the apoptosis of HepG2 cells by following the manufacturer’s instructions. The cells in FITC-positive fraction were considered apoptotic. For ROS, 2 × 10⁵ HepG2 cells were seeded in 12-well plates. After stimulation, dichloro-dihydro-fluorescein diacetate (Applygen Technologies, Beijing, China) were used to incubated cells at 37 °C for 30 min in the dark. Analyses were performed with a FACScan-420 flow cytometry instrument (Becton-Dickinson, USA).

An Annexin V FITC Apoptosis Detection Kit (AD10, Dojindo) was used to evaluate cell apoptosis. Briefly, cells exposed to different treatments were resuspended in diluted Annexin V binding solution to a final cell concentration of 1 × 10⁵ cells/100 µL. Then, the cells were incubated with 5 µL of FITC-conjugated Annexin V and 5 µL of PI for 15 min at room temperature in the dark. Before flow cytometry, the above solution was diluted to 500 µL with Annexin V binding solution. The percentage of apoptotic cells was calculated as the number of Annexin V(+)/PI(−) cells divided by the number of Annexin V(+)/PI(+) cells.
Cell cycle distribution was evaluated by FCM following PI staining, as previously described^{16 (link)}. PLC/PRF/5 and HLE cells were transfected with si-afp and V-afp and then treated with 10 nM rapamycin for 24 h. A FACScan-420 flow cytometer (Becton-Dickinson, USA) was used to measure cellular DNA content.