Cell viability and apoptosis assay

MHCC97H cells were plated in 96-well plates at 4000 cells/well (n = 6) containing 100 μl of DMEM +10 % FBS treated with 400 μM CoCl₂ and cultured for 24 h, then incubated with sorafenib or metformin for another 48 h. DMEM (100 μl) and CCK8 (10 μl) were added to each well and incubated for 2 h. Then, the absorbance was detected with a microplate reader at a test wavelength of 450 nm.

Annexin V/PI was applied to investigate the impact of sorafenib or metformin on cell apoptosis. After treatments, the cells were added with 5 μl Annexin V and 10 μl PI staining supplied by the Annexin V-FITC Apoptosis Detection Kit (SANGON, Cat. No. BS6336), then the results were measured by flow cytometry using a FACS flow cytometer (Becton Dickinson verse, San Jose, CA).