2.6. Acridine orange/ethidium bromide (AO/EtBr) dual staining assay

The apoptosis stimulation by Rhaponticin on MG-63 cells were examined through dual staining. Briefly, MG-63 cells were grown on a 6-well plate with DMEM medium in a moistened CO₂ (5%) incubator at 37 °C for 24hrs. Cells were then cleansed with buffered saline, and cell nuclei were counterstained with AO/EB mixture (1:1, 100 μg/ml) for 15 min. Lastly, cells were cleaned with saline, inspected, and photographed using a fluorescent microscope. The captured images were analyzed using ImageJ software.