Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry

The aim of this study was to evaluate the ability of dual acridine orange/ethidium bromide (AO/EB) staining to detect tumor cell apoptosis. According to apoptosis-associated changes of cell membranes during the process of apoptosis, a clear distinction is made between normal cells, early and late apoptotic cells, and necrotic cells. We cultured human osteosarcoma cells with 30, 60, and 120 µg/ml kappa-selenocarrageenan. To assess the rates of cell proliferation and apoptosis, cells were fluorescently stained with acridine orange/ethidium bromide (AO/EB) or stained with propidium iodide (PI) and analyzed by flow cytometry. All experiments were repeated at least 3 times. Normal tumor cells, early and late apoptotic cells, and necrotic cells were examined using fluorescent microscopy. Early-stage apoptotic cells were marked by crescent-shaped or granular yellow-green acridine orange nuclear staining. Late-stage apoptotic cells were marked with concentrated and asymmetrically localized orange nuclear ethidium bromide staining. Necrotic cells increased in volume and showed uneven orange-red fluorescence at their periphery. Cells appeared to be in the process of disintegrating. The percentage of apoptotic osteosarcoma cells detected by dual acridine orange/ethidium bromide (AO/EB) staining was not significantly different from that detected using flow cytometry (P>0.05). Our results suggest that dual acridine orange/ethidium bromide staining is an economic and convenient method to detect apoptosis in tumor cells and to test tumor chemosensitivity compared with flow cytometry.