DETECTION AND QUANTIFICATION OF LIVE, APOPTOTIC, AND NECROTIC HUMAN PERIPHERAL LYMPHOCYTES BY SINGLE-LASER FLOW-CYTOMETRY

Regulation of peripheral lymphocyte number involves a poorly understoo d balance between cell renewal and loss, Disrupting this balance leads to a large number of disease states. Methods which allow-qualitative and quantitative measurements of cell viability are increasingly valua ble to studies directed at revealing the mechanisms underlying apoptot ic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the re lative number of live, apoptotic, and late-stage apoptotic and necroti c peripheral lymphocytes, Following in vitro gamma irradiation and sta ining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy, This flow c ytometric method is directly compared with the techniques of trypan bl ue exclusion and DNA fragmentation to quantify cell death following ex posure to various doses of in vitro gamma irradiation and postirradiat ion incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic periphera l blood mononuclear cells (PBMC); similar fluorescent patterns are sho wn for radiation- and costicosteroid-treated murine thymocytes; activa ted human PBMC; and PBMC from human immunodeficiency virus-infected in dividuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is o verall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods teste d. By using standard laser and filter settings commonly available to f low cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.