A rapid single-laser flow cytometric method for discrimination of early apoptotic cells in a heterogenous cell population O LIVIER H ERAULT, 1,2 P HILIPPE C OLOMBAT, 1,3 J ORGE D OMENECH , 1,2 MICHEL D EGENNE , 2 J EAN-L OUIS B REMOND, 2 L UC S ENSEBE , 4 MARIE -C HRISTINE B ERNARD 1 AND C HRISTIAN B INET 1,2 1 JE-1993, Faculty of Medicine, Tours, 2 Laboratory of Haematology and 3 Department of Haematology/Oncology, University Hospital, Tours, and 4 Regional Blood Bank, Tours, France Received 9 September 1998; accepted for publication 16 November 1998 Summary. 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 þ annexin V-FITC þ cells), apoptotic (7-AAD – annexin V-FITC þ cells) and viable cells (7-AAD ¹ annexin V-FITC – cells) in a subset of PE þ 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 þ cells as an example of its application. Keywords: flow cytometry, apoptosis, 7-amino-actino- mycinD, annexinV, heterogenous cell population. Apoptosis is an important mechanism for the selective elimination of mammalian cells which is distinct from the process of cell death by necrosis (Wyllie et al, 1984). A recently reported flow cytometric method (Vermes et al, 1995) enables discrimination of these two cell death processes using annexin V and propidium iodide (PI). Nevertheless it does not permit the study of a subset of a heterogenous cell population with a single laser. The anticoagulant annexin V is a member of a family of proteins that are structurally related and exhibit Ca 2þ - dependent phospholipid-binding properties. Annexin V can bind to various phospholipid species and shows its highest affinity for phosphatidylserine (Andree et al, 1990). In normal blood cell phosphatidylserine is situated on the inner layer of the plasma membrane. When cell death occurs, phosphatidylserine is translocated to the outer layer of the membrane, i.e. the external surface of the cell (Fadok et al, 1992). This occurs in the early phase of apoptosis during which the cell membrane itself remains intact. Thus, fluorescein isothiocyanate (FITC)-labelled annexin V is usually used for the quantification of apoptotic cells (Homburg et al, 1995; Koopman et al, 1994). Necrosis is a form of cell death that differs from apoptosis (Kerr et al, 1972; Majno & Joris, 1995; Wyllie et al, 1984) and includes the loss of cell membrane integrity. Measurement of annexin V binding, performed simultaneously with a dye exclusion test, is commonly used to detect early apoptotic cells (Vermes et al, 1995). The exclusion test is performed with fluorescent nonvital dyes forming fluorescent complexes with the DNA which emit fluorescence in the orange range of the spectrum. PI has been widely utilized in assays using annexin V FITC. Never- theless, the brightness of its staining and its extensive spectral emission overlap with phycoerythrin (PE) (Schmid British Journal of Haematology , 1999, 104, 530–537 530  1999 Blackwell Science Ltd Correspondence: Dr O. He ´rault, Laboratoire d’He ´matologie, JE 1993, Faculte ´ de Me ´decine, 2 bis boulevard Tonnelle ´, 37032 Tours Ce ´dex, France.