FLOW CYTOMETRIC DETERMINATION OF APOPTOSIS IN HETEROGENEOUS CELL-POPULATIONS

We have shown that apoptosis and surface antigen expression can be detected simultaneously using multicolor flow cytometry. Apoptosis was measured with an in situ assay that makes use of the ability of the enzyme terminal deoxyribonucleotidyl transferase to catalyze the addition of biotinylated nucleotides to the free 3'-OH groups produced during endonucleolytic cleavage of DNA. The incorporation of the biotinylated nucleotides was quantified using fluorochrome-coupled streptavidin. Immunofluorescence was used to identify the phenotype of apoptotic cells using three color flow cytometry. In model systems, apoptosis was detected in human PBMNC treated with the DNA topoisomerase I inhibitor CAM and in murine thymocytes treated with DEX. The simultaneous application of this method for detecting apoptosis and immunofluorescence offers several advantages. (1) Apoptotic and necrotic cells can be distinguished. (2) The phenotype of the cells undergoing apoptosis can be determined in heterogeneous systems. Thus, we could show that T cells are relatively resistant to CAM-induced apoptosis compared to other peripheral blood mononuclear cells and that both double negative and single positive thymocytes are resistant to steroid-induced apoptosis. (3) The flow cytometric TdT assay to detect apoptosis-associated DNA degradation method is extremely sensitive compared with gel electrophoresis. As few as 2-5 x 10(3) cells give an adequate signal and it is possible to detect DNA strand breaks even when only a small proportion of the cells are undergoing apoptosis. Neither the sensitivity nor specificity of this method can be matched by any electrophoretic method of detecting apoptosis.