Journal of Immunologwal Methods, 139 (1991) 271-279 © 1991 ElsexaerScience Pubhshers B V 0022-1759/91/$03 50 ADONIS 0022175991001841 271 JIM05939 A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry I. Nicoletti 1, G. Migliorati 2, M.C. Pagliacci 1, F. Grignani 1 and C. Riccardi 2 1 Istttuto dt Chmca Medwa 1, and ' lstttuto dl Farmacologta, Perugla Umverstty School of Medicine, 06100 Perugla, Italy (Recewed 19 October 1990, rewsed received 14 January 1991, accepted 19 February 1991) Corticosteroids, calcium ionophores and anti-CD3 monoclonal antibodies kill mouse thymocytes incubated in vitro. Cell death is preceded by extensive DNA fragmentation into ohgonucleosomal subumts. Tins type of cell death (apoptosis), which physiologically occurs m the intrathymic process of immune cell selection, is usually evaluated by either electrophoretic or colorimetric methods which measure DNA fragmentation in the nuclear extracts. These techniques are unable to determine the percentage of apoptotic nuclei or recognize the apoptotic cells in a heterogeneous cell population. We have developed a flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer and have compared it with the classical colonmetric and electrophoretic techniques using dexamethasone (DEX)-treated mouse thymocytes. Apoptotic nuclei appeared as a broad hypodiploxd DNA peak which was easily discriminable from the narrow peak of thymocytes with normal (diploid) DNA content in the red fluorescence channels. When the DEX-induced apoptosis was inhibited by either low-temperature (4°C) incubation or cyclohexlmide treatment, no hypodiploid DNA peak appeared. Similarly, thymocyte death induced by sodmm azide, a substance with cell-kilhng activity through non-apoptotic mechanisms, &d not result in any variation in the normal DNA peak. The flow cytometrlc data showed an excellent correlatton with the results obtained with both electrophoret~c and colorimetric methods. This new rapid, simple and reproducible method should prove useful for assessing apoptos~s of specific cell populations in heterogeneous tissues such as bone marrow, thymus and lymph nodes. Key words Apoptosls, Thymocyte, DNA fragmentation, Flow cytometry, Dexamethasone Introduction Apoptosis is a common form of cell death xn eukariotes. The process is operative during em- bryogenesis, in tumor regression and in the Correspondence to I. Ntcoletti, IsUtuto dt Chmca Med~ca1, Umversit/t dl Perugia, Pohchmco Monteluce, 06100 Perugaa, Italy elirmnation of self-reactive T lymphocytes m the thymus (Wyllie et al., 1980). Apoptotic cell death can be induced by glucocorticoid treatment (Cohen and Duke, 1984), antibodies to CD3/T cell recep- tor complex (Shi et al., 1989; Smith et al., 1989), exposure to Ca 2+ lonophores (Kazaki et al., 1989; McConkey et al., 1989) or "y-irradiauon of mouse thymocytes (Selllns and Cohen, 1987), anti-APO monoclonal antibody treatment of tumor cells (Trauth et al., 1989) and growth factor deprivation