Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fcγ and complement receptors.

INTRODUCTION:This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. METHODS:Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. RESULTS:The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 μg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. DISCUSSION:The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.