A rapid single-laser flow cytometric method for discrimination of early apoptotic cells in a heterogenous cell...

A recently reported cytometric method described the possibility of discriminating apoptotic from necrotic cells using FITC-labelled annexin V and propidium iodide (PI). Nevertheless, the brightness of PI-staining and its extensive spectral emission overlap with phycoerythrin (PE) does not permit the study of a subset of a heterogenous cell population with single laser instrumentation. The surface staining of a subset with PE in a heterogenous cell population therefore requires another exclusion dye to detect necrotic cells. We used 7-amino-actinomycin D (7-AAD) that can be excited by the 488 nm argon laser line. 7-AAD emits in the far red range of the spectrum and 7-AAD spectral emission can be separated from the emissions of FITC and PE. The fluorescence parameters allow characterization of necrotic (7-AAD+ annexin V-FITC+ cells), apoptotic (7-AAD-annexin V-FITC+ cells) and viable cells (7-AAD- annexin V-FITC- cells) in a subset of PE+ cells. The value of this method was demonstrated by measuring apoptosis and necrosis in a model of HL-60 cells exposed to different inducers of cell death. The method was validated by fluorescent cell sorting in combination with morphologic examination of the sorted cells. The technique we present is particularly valuable in a clinical setting because it enables rapid multiparameter analysis of necrosis and early apoptosis in combination with cell surface phenotyping with a single laser. We present the effects of haemopoietic growth factor deprivation on myeloid progenitor CD34+ cells as an example of its application.