SARS-CoV-2 RBD Opsonization and Phagocytic Uptake by THP-1 Cells

The THP-1 (TIB-202, ATCC) ADCP with antigen-coated beads was conducted as described (26 (link)). SARS-CoV-2 RBD (BEI Resources NR-52309) was biotinylated with Sulfo-NHS-LC Biotin (Thermo Fisher), then incubated with 1μm fluorescent neutravidin beads (Invitrogen) at 4°C for 16hours. Excess antigen was washed away and RBD-coupled neutravidin beads were resuspended in PBS-0.1% bovine serum albumin (BSA). RBD-coupled beads were incubated with serially diluted samples (1:100, 1:500, 1:2500) in duplicate for 2hours at 37°C. THP1 cells (1×10⁵ per well) were then added. Serum opsonized RBD-coupled beads and THP1 cells were incubated at 37°C for 16hours, washed and fixed with 4% PFA. Bead uptake was measured on a BD LSRFortessa and analyzed by FlowJo10. Phagocytic scores were calculated as the integrated median fluorescence intensity (MFI) (%bead-positive frequency×MFI/10,000) (131 (link)). The background signal (PBS) was subtracted. Experiments were conducted two independent times. Representative data from one dilution was chosen by the highest signal-to-noise ratio.