Studies on the binding of C3b-coated microspheres to human neutrophils

A method is described for the quantitation of C3b receptors on human neutrophils using a mixture of C3b-coated fluorescent and C3b-coated non-fluorescent microspheres. The method measures the "sterically available' C3b receptors on the cells, for example, the receptors available to opsonized bacteria. The use of mixtures of fluorescent and non-fluorescent microspheres resulted in lowered fluorescence intensities of the microsphere-coated neutrophils that were well within the fluorescence limitations of fluorescence activated cell analyzers or sorters used in the assay procedure. These mixtures also allowed the distribution of the C3b-coated microspheres around the neutrophils to be easily visualized in the fluorescence microscope. The binding of the C3b-coated microspheres to the neutrophils was shown to be receptor mediated by typical saturable binding kinetics, by complete inhibition by fluid phase C3b, but not by other proteins and by nearly complete inhibition by anti-C3b receptor antibody. Several parameters that could affect the binding of C3b-coated microspheres to neutrophils were studied; these included time and temperature of incubation of the microspheres with the cells, the diameter of the microspheres, the C3b content of the C3b-coated microspheres, the presence of metal ions, azide, EDTA, protein (BSA, IgG), soybean trypsin inhibitor in the buffers, and the method of isolation of the neutrophils. The C3b-coated microspheres were evenly distributed around the neutrophils in almost all of the cases; however, the neutrophils used in these studies were not activated and were not phagocytosing. The method is extremely reproducible and sensitive in detecting small changes in number of C3b receptors on cells.