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

A recently reported cytometric method described the possibility of discrimi nating apoptotic from necrotic cells using FITC-labelled annexin V and prop idium iodide (PI). Nevertheless, the brightness of PI-staining and its exte nsive spectral emission overlap with phycoerythrin (PE) does not permit the study of a subset of a heterogenous cell population with single laser inst rumentation. The surface staining of a subset with PE in a heterogenous cel l population therefore requires another exclusion dye to detect necrotic ce lls, We used 7-amino-actinomycin D (7-AAD) that can be excited by the 488 n m 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. T he fluorescence parameters allow characterization of necrotic (7-AAD(+) ann exin 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 mo del of HL-60 cells exposed to different inducers of cell death. The method was validated by fluorescent cell sorting in combination with morphologic e xamination of the sorted cells. The technique we present is particularly va luable in a clinical setting because it enables rapid multiparameter analys is of necrosis and early apoptosis in combination with cell surface phenoty ping with a single laser. We present the effects of haemopoietic growth fac tor deprivation on myeloid progenitor CD34(+) cells as an example of its ap plication.