Discrimination of viable and non‐viable cells using propidium iodide in two color immunofluorescence

The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein- and phycoerythrin-conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics. We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.