Annexin V-PI double staining assay

HepG2, Huh7, or primary liver cancer cells were collected and washed in cold PBS. Annexin-binding buffer (1X) was prepared, then 5 μl of the 1 mg/ml PI solution was diluted in 45 μl 1X annexin-binding buffer. Cells were re-suspended in 1X annexin-binding buffer to 1 × 10⁶ cells/ml. FITC annexin V (5 μl) and PI solution (1 μl) were added to 100 μl cell suspension for 15 min at room temperature (25°C). Then, 400 μl 1X annexin-binding buffer was added, gently mixed, and the mixtures were kept on ice. The stained cells were analyzed by flow cytometry.