RAPID QUANTIFICATION OF LYMPHOCYTE SUBSETS IN HETEROGENEOUS CELL-POPULATIONS BY FLOW-CYTOMETRY

Determination of the number of viable cells or quantification of lymph ocyte subsets in heterogeneous cell populations is critically importan t for cytotoxicity assays, apoptosis assays, or the analysis of differ ential activation of T-cell subsets by distinct stimuli. In this repor t, we describe a rapid flow cytometry method termed Standard Cell Dilu tion Analysis (SCDA) specifically to quantify any subset of phenotypic ally definable, viable cells in heterogeneous populations using a FACS can flow cytometer. This method combines: (1) specific detection of ly mphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2 ) electronic exclusion of dead cells or cell debris by propidium-iodid e staining and gating on forward vs. sidescatter, respectively, and (3 ) admixture of a known amount of fixed, fluorescein isothiocyanate sta ined cells immediately before analysis as a constant parameter to allo w for calculation of cell quantity. We have used SCDA to analyze the i n vitro growth characteristics of various human T-lymphocyte subpopula tions in response to different activation stimuli. (C) 1994 Wiley-Liss , Inc.