Antibody-Dependent Cellular Phagocytosis and Antibody-Dependent Neutrophil Phagocytosis Assays

ADCP and ADNP were conducted as previously described [80 (link),81 (link)]. Briefly, spike or RBD proteins were biotinylated using EDC (Thermo Fisher Scientific) and EZ-link Sulfo-NHS-LC-LC (Thermo Fisher Scientific) and then coupled to yellow/green and then coupled to yellow/green FluoSphere NeutrAvidin-conjugated beads (Thermo Fisher Scientific, F8776). Immune complexes were formed by incubating the bead+protein conjugates with diluted serum (ADNP 1:50 dilution, ADCP 1:100 dilution) for 2 hours at 37°C and then washed to remove unbound antibody. The immune complexes were then incubated overnight with THP-1 cells (ADCP), or for 1 hour with RBC-lyzed whole blood (ADNP). THP-1 cells were then washed and fixed in 4% PFA, while the RBC-lyzed whole blood was washed, stained for CD66b Pacific Blue (BioLegend - San Diego, California, USA), CD3-AlexaFluro700 (BD Biosciences - Miami, Florida, USA), and CD14-APC-Cy7 (BD Biosciences) to identify neutrophils (CD3- CD14- CD66b+) and then fixed in 4% PFA. Flow cytometry was performed to identify the percentage of quantity of beads phagocytosed by THP-1 cells or neutrophils, and a phagocytosis score was calculated (% cells positive × median fluorescent intensity of positive cells). Flow cytometry was performed with an LSRII (BD), and analysis was performed using FlowJo V10.7.1.