A RAPID METHOD FOR MEASURING APOPTOSIS AND DUAL-COLOR IMMUNOFLUORESCENCE BY SINGLE LASER FLOW-CYTOMETRY

A sensitive method for quantification of cells undergoing apoptosis th at permits the simultaneous measurement of dual-color cell surface imm unofluorescence is presented. Unfixed cells are stained with 7-amino-a ctinomycin D (7-AAD) for discrimination of live from early apoptotic c ells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). Owing to its spectral characteristics 7-AAD can be combined with fluorescein-isothiocyanate (FITC) and phycoeryth rin (PE) cell surface staining. After staining, the samples can be tre ated with paraformaldehyde (PF) solution to eliminate the risk for exp osure of laboratory personnel to biohazardous agents and to preserve t he cells through fixation for later analysis on the flow cytometer. Th e value of the method is shown on the measurement of apoptosis in huma n thymocytes and in human peripheral blood mononuclear cells (PBMC) ex posed to various inducers of active cell death. The method is validate d by fluorescent activated cell sorting in combination with morphologi c examination of the sorted cells. The technique we are presenting is particularly valuable in a clinical setting because it allows rapid mu ltiparameter analysis of apoptosis in combination with cell surface ph enotype on biohazardous samples with single laser instrumentation.