A NEW FLOW CYTOMETRIC METHOD FOR QUANTITATIVE ASSESSMENT OF LYMPHOCYTE MITOGENIC POTENTIALS

A new flow cytometric method was developed to quantitatively assess ly mphocyte proliferation simultaneously for different subsets. The cells were stained with a fluorescent dye, PKH-26 and were stimulated with mitogens. The fluorescence intensities (FL2) of proliferating cells we re measured by flow cytometry; and each subset was identified by the u se of a monoclonal antibody (Mab)-fluorescein-isothiocyanate (FITC) (F L1). FL2 histograms were then analyzed by the cell proliferation model based on the ModFit software (Verity). This new method revealed infor mation which could not be obtained by conventional mitogen assays. For example, the CD4(+) and the CD4(-) T-subsets responded to phytohemagg lutinin (PHA) quite differently from each other and it was indicated t hat activation of one population could significantly alter the respons e of the other. In addition, even within a subset, all activated cells did not proliferate uniformly. Some cells divided only once while oth ers underwent further cellular division during the same time period. T he method is, therefore, invaluable for studying the nature and the ex tent of interactions between different cellular subsets within a cultu re.