New, simple flow cytometry technique to discriminate between internalized and membrane‐bound particles in phagocytosis

Abstract

This report describes a new flow cytometry technique to measure phagocytic activity and discriminate simultaneously between internalized and membranebound particles. Fluorescein‐conjugated heat‐killed Candida albicans (F‐Ca) are opsonized with purified antibodies or normal human serum and used as targets for human polymorphonuclear granulocytes (PMN). The procedure is based on the observation that F‐Ca lose their green fluorescence and acquire red fluorescence upon incubation with ethidium bromide (EB) through the resonance energy‐transfer phenomenon occurring between the two fluorochromes. PMN are incubated with opsonized F‐Ca particles for 20 min at 37°C or, as a control, at 4°C and in the presence of cytochalasin B, an inhibitor of the phagocytic process that does not affect membrane binding of F‐Ca. EB is added, and green and red fluorescence associated with PMN is evaluated using a mercury‐lamp‐powered instrument. Because EB does not penetrate intact cell membranes, internalized particles are not affected by EB and remain green, whereas membrane‐bound particles assume an intense red stain. By means of contour plot analysis, the number of PMN containing and/or binding F‐Ca particles can be readily assessed. The method described here allows precise quantitative analysis of the phagocytic process on the part of human PMN in a single, one‐step assay that does not require sophisticated instrumentation or reagents and should prove to become a test suitable for clinical purposes. Copyright © 1989 Wiley‐Liss, Inc.