Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry

Determination of the number of viable cells or quantification of lymphocyte subsets in heterogeneous cell populations is critically important for cytotoxicity assays, apoptosis assays, or the analysis of differential activation of T-cell subsets by distinct stimuli. In this report, we describe a rapid flow cytometry method termed Standard Cell Dilution Analysis (SCDA) specifically to quantify any subset of phenotypically definable, viable cells in heterogeneous populations using a FACScan flow cytometer. This method combines: (1) specific detection of lymphocyte subsets by phycoerythrin-conjugated monoclonal antibodies, (2) electronic exclusion of dead cells or cell debris by propidium-iodide staining and gating on forward vs. sidescatter, respectively, and (3) admixture of a known amount of fixed, fluorescein isothiocyanate stained cells immediately before analysis as a constant parameter to allow for calculation of cell quantity. We have used SCDA to analyze the in vitro growth characteristics of various human T-lymphocyte subpopulations in response to different activation stimuli.