1μm yellow fluorescent neutravidin beads

Monocyte mediated phagocytosis was assessed with a bead-based assay using the THP-1 cell line, as previously described [49 (link)]. Briefly, biotinylated (EZ-Link Sulfo-NHS-LC-LC-Biotin, Thermo Fisher) gp140 ConS (Duke University) antigen was coupled to 1μm yellow fluorescent neutravidin beads (Thermo Fisher) for 2 h at 37°C. After removal of excess antigen by washing with 0.1% Bovine serum albumin (BSA) in phosphate buffered saline (PBS) for blocking, saturated beads (1.82×10⁸ beads/well) were incubated with samples at a 1:100 dilution in PBS for 2 h at 37°C. Immune complexes were washed and 2.5×10⁴ THP-1 cells (American Type Culture Collection) were added per well and incubated for 16 h at 37°C. Cells were fixed in 4% paraformaldehyde (PFA) and sample acquisition was performed via flow cytometry (IntelliCyt, iQue Screener plus). Events were gated on single cells and bead positive cells. A phagocytosis score was calculated as the percent of bead positive cells× GMFI/1,000. All samples were run in duplicate on separate days.

A bead-based assay was used to assess the ADCP by monocytes. The THP-1 based phagocytosis assay was performed as previously described (30 (link)). Briefly, avi-tagged biotinylated (Avidity, BirA500) gp120 MN used in the RV144 boost (Duke University) antigen was coupled to 1 μm yellow fluorescent neutravidin beads (Thermo Fisher Scientific) for 2 hours at 37°C. After removal of excess antigen by washing with 0.1% Bovine serum albumin (BSA) in phosphate buffered saline (PBS) for blocking, saturated beads (1.82 × 10⁸ beads/well) were incubated with the appropriately diluted sample (3 dilutions at 1:100, 1:500, and 1:2500) in order to calculate the AUC. Immune complexes were washed, and 2.5 × 10⁴ THP-1 cells (American Type Culture Collection) were added per well and incubated for 16 hours at 37°C. Cells were fixed in 4% PFA, and sample acquisition was performed via flow cytometry (Stratedigm, S1000EXi). Events were gated on single cells and bead-positive cells; a phagocytosis score was calculated by the percentage of bead-positive cells GMFI/10,000. The AUC was calculated using GraphPad Prism based on 3 dilutions and a duplicate.