Yellow fluorescent neutravidin beads

Monocyte-mediated phagocytosis was measured with a bead-based assay and the THP-1 cell line, as previously described^{64 (link)}. Briefly, biotinylated (EZ-Link Sulfo-NHS-LC-LC-Biotin, Thermo Fisher) BG505 SOSIP trimer was coupled to 1 μm yellow fluorescent neutravidin beads (Thermo Fisher) for 2 h at 37 °C. Excess antigen was removed in two washes with 0.1% BSA in PBS for blocking. Then, saturated beads (1.82 × 10⁸ beads/well) were incubated with 10 μL of mAb (RM19R, VRC01, or PGT145 at various concentrations, starting at 5 μg/mL, 2-fold dilutions) in PBS for 2 h at 37 °C. ICs 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/1000. All samples were run in duplicate.

Antibody-dependent cellular phagocytosis (ADCP) was assessed in a bead-based assay (25 (link)). Antigens were biotinylated using Sulfo-NHS-LC-LC Biotin for 2h at room temperature (Thermo Scientific) and excess biotin was removed with a 3-kDa molecular mass cutoff column (Amicon/EMD, UFC500396). Yellow, fluorescent neutravidin beads (Thermo Fisher Scientific) were coupled to biotinylated ESAT-6/CFP-10, PPD and LAM for 2 h at 37°C. Subsequently, beads were washed and blocked with 1% BSA for 1h at room temperature. Then, antigen-coupled beads were incubated with 1:30 diluted plasma in PBS for 2 hours at 37°C. Following immune complex formation, samples were washed and 2.5x10⁴ THP-1 cells (ATCC) were added per well and incubated for 16 hours at 37°C in RPMI media with beta-mercaptoethanol. Following fixation, the next morning, sample acquisition was performed via flow cytometry (Intellicyt, iQue Screener plus) utilizing a robot arm (PAA), and analysis occurred using Forecyt software. Gating strategy included gating on THP-1 cells, single cells and FITC-positive events. A phagocytosis score was calculated as (percentage of bead-positive cells) x (MFI of bead-positive cells) divided by 10,000.