Acridine orange/propidium iodide staining (AO/PI)

After 24 h incubation of 1 × 10⁶ cells/well of HepG2 cells in 6-well plates at 37 °C in a humidified CO₂ incubator, treatment and control groups treated with 150 μg/ml ZnO NPs and 0.1% DMSO, respectively, for three timepoints (24, 48, and 72 h). Then, the cells were separated with trypsin and AO/PI dyes were added to suspension cells at an equal ratio (10 μl) and were examined under fluorescent microscope. All viable and early apoptotic cells only uptake the AO dye which bind to double-strand DNA and emit green fluorescence, while PI stained the necrotic and dead cells and look red.