Ž . Journal of Immunological Methods 209 1997 111–123 Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods Herve Lecoeur, Eric Ledru, Marie-Christine Prevost, Marie-Lise Gougeon ) ´ ´ Unite d’Oncologie Virale, Departement SIDA et RetroÕirus, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France ´ ´ ´ Received 22 May 1997; revised 10 July 1997; accepted 12 August 1997 Abstract The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC–annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSCrSSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSCrSSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD low with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debrisrapoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSCrSSC criteria and 7-AADrFSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily Abbreviations: PCD, Programmed cell death; PBMC, Peripheral blood mononuclear cells; 7-AAD, 7-Amino actinomycin D; dUTP, Deoxy-uridine tri-phosphate; ISNT, In situ nick translation; FSC, Forward scatter; SSC, Side scatter; EtOH, ethanol; PS, Phosphatidyl serine; ASX, Asymptomatic; AIDS, Acquired immune deficiency syndrome; HIV, Human immunodeficiency virus ) Corresponding author. Tel.: q33-1-45688907; fax: q33-1-45688909; e-mail: mlgougeo@pasteur.fr 0022-1759r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0022-1759 97 00138-5