Functional heterogeneity of human monocytes--with a special reference to flow cytometric assays

A large number of human mononuclear cells were simultaneously separated into fractions enriched in B cells, T cells, large granular lymphocytes (LGL) and monocytes by centrifugal elutriation. Lymphocyte populations were analyzed using monoclonal antibodies. In particular, highly enriched natural killer cells, Leu7+ cells, were collected in the intermediate fractions. Monocytes, which were identified as esterase positive cells, and Leu M3 cells were collected at higher counterflow rates and in the final fraction. The purity of monocytes in the final fraction was 81%. The oxidative metabolic activity (H2O2 production) and non-specific esterase activity of individual monocytes was estimated in the analysis of functional heterogeneity of monocytes using flow cytometry. 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescein diacetate (FDA) were used as indicators in the measurement of H2O2 generation and esterase activity. Intracellular generation of a fluorescence product (H2O2 Production; average percentage of fluorescence positive cells) of monocytes in the stimulation of phorbol myristate acetate (PMA, 100 ng/ml) was greater in larger than smaller cells. H2O2 production gradually increased from 6% and 25-38% and 60% in the intermediate and final fractions respectively. Furthermore, the average fluorescence intensity of the large monocyte population in the final fraction was 1.13-1.31 fold more active than that of the smaller cells. Thus, the functional heterogeneity of human monocytes was further confirmed in the assays of H2O2 production exposed to PMA and FDA hydrolysis using flow cytometry. Furthermore, the CCE system can isolate lymphocyte subsets and LGL.