Characterization of human mononuclear cells using reduced pyridine nucleotide fluorescence and flow cytometry

Peripheral blood monocytes undergo an oxidative burst similar to that seen in neutrophils. The basis for this response appears to be an NAD(P)H oxidase that utilizes reduced NAD(P)H to form superoxide anion. We utilized the unique UV-stimulated fluorescence property of reduced pyridine nucleotides to analyze NAD(P)H utilization in monocytes. UV-stimulated fluorescence in mononuclear cell preparations indicated two populations of cells with the highly fluorescent cells having a Coulter volume consistent with that of monocytes. Dual laser analysis with monoclonal antibodies confirmed that these highly fluorescent cells are monocytes by showing them to be OKM1+, Leu DR+, and anti-monocyte 0.2+. Natural killer (NK) cells, as defined by Leu 7, were not found in this highly fluorescent population. Stimulation of mononuclear cells with phorbol myristate acetate caused a fluorescence loss indicative of NAD(P)H oxidation in monocytes but not in lymphocytes. Stimulation with suboptimal concentrations of PMA (1-5 ng/ml) resulted in a dose-dependent fluorescence loss in monocytes that occurred in an all-or-none fashion identical to the pattern observed in neutrophils. Simultaneous measurement of H2O2 production using dichlorofluorescein formation with NAD(P)H fluorescence indicates that oxidant production occurs in a graded manner. This method, then, provides a convenient way to study in single cells the metabolic events involved in depletion and replenishment of NAD(P)H during the oxidative burst and demonstrates an additional means by which to distinguish monocytes from lymphocytes using flow cytometry.