Acridine orange/ethidium bromide (AO/EtBr) double staining

The AO/EtBr dual staining technique was performed as described by Liu et al.⁹ Briefly, cells were seeded in a 96-well plate at a density of approximately 10⁴ cells/well. Following incubation with betahistine for 48 hours, cells were trypsinized, and 10-25 µL cell suspensions were transferred onto glass slides. One microliter of AO/EtBr stain­ing solution (a mixture of dyes containing 100 µg/mL AO and 100 µg/mL EtBr) was added to cell suspensions, and then the samples were covered with a coverslip. The cell morphology was examined under a fluorescent microscope (Carl-Zeiss/Axio observer 3., Zen 2.3 Blue Edition software) within 20 minutes after addition of the Ao/EtBr stain. For statistical analysis, at least 200 cells were counted, and the results were expressed as mean values obtained from at least three independent experiments. In the asay, both live and dead cells are stained with AO, while EtBr stains only dead cells that have lost membrane integrity. Live cells appear uniformly green, whereas early apoptotic cells show green dots in their nuclei. Late apoptotic cells stain orange and show condensed and/or often fragmented nuclei. Necrotic cells stain orange, with a nuclear morphology resembling that of viable cells, but without condensed chromatin.⁹