Cell cycle and apoptosis analysis

Cell cycle and apoptosis analysis were performed by flow cytometry using Cell Cycle Assay kit-PI/RNase Staining and Annexin V/FITC Apoptosis Detection kit (Roche, Basel, Switzerland) according to manufacturer’s instructions. Cell cycle analysis was carried out by Cell Cycle Assay Kit-PI/RNase Staining. Briefly, the cell density was adjusted to 2×10 ⁶ cells/mL, and 1 mL of cell suspension was added into a 1.5-mL microtube and centrifuged at 1000 g for 3 min. Then the supernatant was discarded and 1 mL of 70% ethanol (–20°C) was added to the cell pellet to disperse the cells by votexing and standing at 4°C for 2 h. After centrifugation at 1000 g for 3 min, the ethanol was removed and 1 mL PBS buffer was added to wash the cells. After centrifugation at 1000 g for 3 min, the supernatant was discard and 0.5 mL of Working Solution (500 μL assay buffer, containing 25 μL PI solution and 2.5 μL RNase solution) was added. After vortexing and incubation for 30 min at 37°C in the dark, the mixture was further incubated for 30 min at 4°C in the dark, followed by vortexing and filtering through a nylon mesh to remove cell clumps in the sample. Finally, cell cycle was detected by flow cytometry.

Cell apoptosis analysis was carried out using Annexin V/FITC Apoptosis Detection kit. Briefly, HCC cells (treated as above) were harvested by trypsinization, rinsed with ice-cold phosphate PBS, and centrifuged to remove the supernatant. Then, the cells were resuspended in 100 μL 1× binding buffer and incubated with Annexin V-FITC for 15 min in the dark at room temperature. Finally, a flow cytometer was used to determine the number of apoptotic cells from B2 and B4 quadrants.