SENSITIVE METHOD FOR MEASURING APOPTOSIS AND CELL-SURFACE PHENOTYPE IN HUMAN THYMOCYTES BY FLOW-CYTOMETRY

A rapid, gentle, and sensitive method for quantification of cells unde rgoing apoptosis is presented. The method allows the simultaneous dete rmination of dual-color cell surface immunofluorescence. Cells are sta ined for 7 min with the vital dye Hoechst 33342 (H0342) for identifica tion of live and apoptotic cells. 7-amino-actinomycin D (7-AAD) is add ed to distinguish cells that have lost membrane integrity from apoptot ic and live cells. Due to its spectral properties 7-AAD can be utilize d on cells that are dual-surface labelled with fluoresceinisothiocyana te (FITC) and phycoerythrin (PE). The value of the method is demonstra ted on human thymocytes, which constitutively undergo programmed cell death and which show an increase in the rate of apoptosis after exposu re to the glucocorticoid dexamethasone (DEX). Vital staining with H034 2 permits earlier detection of apoptotic changes compared to a stainin g technique in which cells are treated with a hypotonic citrate soluti on containing propidium iodide (PI) and the apoptotic cells are repres ented in a hypodiploid, ''sub-G(1)'' peak. The H0342/7-AAD method may be particularly applicable to studies of programmed cell death in cell s in which DNA fragmentation is difficult to detect by decreased DNA s tainability. (C) 1994 Wiley-Liss, Inc.