A THP-1 antibody-dependent cellular phagocytosis (ADCP) assay was performed as previously described (55 (link)). Briefly, recombinant HIV proteins were conjugated onto 1-μm fluorescent neutravidin beads (Invitrogen). Excess antigen was removed by washing pelleted beads, which were then incubated with purified IgG antibody samples (100 μg/ml) for 2 h at 37°C. Following opsonization, THP-1 cells were added to the bead-antibody mix and incubated overnight, followed by fixation and analysis of bead uptake by flow cytometry.
1 μm fluorescent neutravidin beads
Manufactured by Thermo Fisher Scientific
1-μm fluorescent neutravidin beads are microscopic particles coated with the protein neutravidin, which has a high affinity for biotin. These beads are fluorescently labeled, allowing them to be easily detected and tracked using fluorescence microscopy techniques.
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4 protocols using 1 μm fluorescent neutravidin beads
1
HIV Protein Bead-Based ADCP Assay
2
SARS-CoV-2 RBD Opsonization and Phagocytic Uptake by THP-1 Cells
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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.
3
Antigen-Specific Phagocytosis Assay for THP-1 Cells
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THP-1 cell phagocytosis of antigen-coated beads was conducted as previously described17 (link),60 (link). Mtb antigens were biotinylated with Sulfo-NHS-LC Biotin (ThermoFisher) following the manufacturer’s instructions and incubated with 1-μm fluorescent neutravidin beads (Invitrogen) at 4 °C for 16 h. Excess antigen was washed away. Antigen-coated beads were incubated with plasma (at 1:100, 1:1,000, 1:10,000 dilutions in PBS) for 2 h at 37 °C. THP-1 cells (1 × 105 per well) were added and incubated at 37 °C for 16 h. Bead uptake was measured in fixed cells using flow cytometry on a BD LSRII (BD Biosciences) equipped with a high-throughput sampler. Phagocytic scores are presented as the integrated MFI (percentage bead-positive frequency × MFI per 10,000) (Extended data Fig. 8a )50 (link). Antibody-dependent cellular phagocytosis experiments for individual plasma samples were performed in duplicate in two independent experiments.
4
Bead-based Fc-Receptor Phagocytosis Assay
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PPD was biotinylated with sulfo-NHS-LC BIOTIN (ThermoFisher) and incubated with 1 μm fluorescent neutravidin beads (Invitrogen) at 4°C for 16 hours. Excess antigen was washed away. Antigen-coated beads were incubated with IgG samples (100 µg/mL) at 37°C for 2 hours to which THP1 cells (1 × 105/well) were added and incubated further at 37°C for 16 hours. Bead uptake in fixed samples was measured on a BD LSRII. The integrated MFI (% bead-positive frequency × MFI/10 000) generated phagocytic scores [30 (link)].
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