Apoptosis detection by acridine orange–ethidium bromide (AO–EB) staining

AO–EB staining was performed to detect the apoptosis (Azizi et al. 2017). AO and EB are DNA-specific dyes which can differentiate dead cells from viable cells. Upon disintegration of the cell membrane, AO can penetrate into both live and dead cells, while EB can intrude into dead cells only. Therefore, AO-stained cells appear green in color (live cells); EB stains dead cells, imparting a greenish yellow to red color during the early and late stages of apoptosis. A549 cells were seeded onto six-well plates at a density of 5000 cells/well and incubated at 37 °C for 24 h under 5% CO₂. ZnO-NPs were added to the cells at their IC ₅₀ concentrations as determined by MTT assay and incubated further for 48 h at 37 °C under 5% CO₂. The spent media were removed, and cells were fixed in chilled methanol at RT for 20 min. Further, methanol was removed and the cells were stained with a mixture of AO and EB stain and incubated at 37 °C in dark for 15 min. The cells were washed with phosphate buffer saline (PBS) to remove the excess stain. Cells were overlaid with PBS (1 ml) and photographed using fluorescent imager (ZOE, BioRad) to detect the nuclear changes.