Flow-cytometry assay for apoptosis using fluorophor 10-N-nonyl acridine orange

Abstract

Recently it has been shown that redox catalytic interactions of cytochrome c with cardiolipin (CL) and subsequent oxidation of CL occurs during apoptosis. Oxidation of CL is accompanied by a release of cytochrome c from the mitochondria into the cytoplasm, a key event in development of apoptosis. 10-N-Nonyl acridine orange (NAO), a fluorophor that forms a stable complex with reduced form of CL, but not with oxidized CL, can be used for flow cytometry analysis of this effect. It has been shown that after the incubation of CTLL-2 cells in the presence of 7% ethanol (90 min) and subsequent staining with NAO, a cell population with low intensity of fluorescence appears. Flow cytometry analysis of the cells by means of a conventional method (annexin V-FITC/propidium iodide (PI)) showed that the cell population with low intensity of NAO fluorescence corresponded to the population of apoptotic cells with good coincidence of percentages. Then apoptosis was induced in the cells of three lines by growth factor deprivation (IL-2-dependent cell line CTLL-2) or as a result of actinomycin D treatment, an RNA synthesis inhibitor (Jurkat and Raji cell lines). Comparison of the data obtained by a conventional assay (annexin V-FITC/PI) and a newly elaborated assay (NAO/PI) has demonstrated a good coincidence. The data obtained with these methods exhibited significant level of correlation (0.953, p < 0.0001).