title: 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 membrane-bound 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 degrees C or, as a control, at 4 degrees 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.
authors.affiliation: Reparto Medicina, D.A.S.R.S., Aeroporto Pratica di Mare, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Fattorossi
authors.forename: R
authors.initials: R
authors.lastname: Nisini
authors.forename: J G
authors.initials: JG
authors.lastname: Pizzolo
authors.forename: R
authors.initials: R
authors.lastname: D'Amelio
mesh_terms: D000328:Adult; D002176:Candida albicans; D003594:Cytoplasmic Granules / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000032:analysis* / Q000502:physiology; D010587:Phagocytosis