Ab-dependent Phagocytosis Assays for Monocytes and Neutrophils

Assays of Ab-dependent phagocytosis by monocytes (ADCP) and neutrophils (ADNP) were performed essentially as described (47 (link)–49 (link)). Briefly, 1 µm yellow-green fluorescent microspheres (Thermo, F8813) were covalently conjugated with recombinant RBD and incubated for 3 h with dilute serum or nasal wash specimens and either the human monocytic THP-1 cell line (ATCC, TIB-202), or with freshly-isolated primary neutrophils. After pelleting, washing, and fixing, phagocytic scores were quantified as the product of the percentage of cells that phagocytosed one or more fluorescent beads and the median fluorescent intensity of this population as measured by flow cytometry with a MACSQuant Analyzer (Miltenyi Biotec). ADCP assays were performed in duplicate with high correspondence between results presented here and the replicate run. ADNP assays were performed in biological replicate using neutrophils purified from two different healthy donors for which results were averaged. A subset of neutrophils was stained with CD66b-APC (BioLegend G10F5) and PI (Biotium 41007) to determine the purity and viability of the isolated cellular fraction. CR3022 and VRC01 were used as positive and negative controls, respectively. Wells containing no Ab were used to define the level of Ab-independent phagocytosis.