FLOW CYTOMETRIC ASSAY FOR PHAGOCYTOSIS OF HUMAN MONOCYTES MEDIATED VIA FC-GAMMA-RECEPTORS AND COMPLEMENT RECEPTOR CR-1 (CD35)

We developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fc-gamma receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA-coated latex beads (B) were incubated with diluted rabbit anti-BSA IgG. To bind complement components, BA-particles were incubated with whole human serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1,4b,2a,3b on BA-particles (BAC1,4b,2a,3b). Further incubation of BAC1,4b,2a,3b with EDTA-GVB at 37-degrees-C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1,4a,2a,3b particles by SDS-PAGE and immunoblot, and these particles were calculated to have approximately 25,000-30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3-mu-M cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1,4b,2a,3b, respectively.