Yellow green carboxylate beads

Antibody-dependent cellular phagocytosis was performed as previously described ^{63 (link)} with slight modifications. Briefly, goat-anti human IgG F(ab’)2 (Invitrogen) was covalently coupled to yellow-green carboxylate beads (Thermofisher). Antibodies were diluted in culture medium to a starting concentration of 133 nM and serially diluted 4-fold 7 times. Diluted mAbs were incubated with anti-human IgG beads for 2 hours at 37°C to form immune complexes. THP-1 (ATCC) cells (25,000/well) were added to the immune complexes and incubated at 37°C for 4 hours. Cells were washed 2x with cold 1x PBS prior to being fixed with 4% paraformaldehyde. The cells were analyzed on a NovoCyte Advanteon flow cytometer (Agilent) (Figure S2C). A phagocytosis score was calculated as the (percentage of FITC+ cells) x (the geometric mean fluorescence intensity (gMFI) of the FITC+ cells)/100,000. Buffer only wells were used as negative controls and the assay was performed in technical replicate with two biological replicates.

Antibody-dependent cellular phagocytosis was performed as previously described⁷⁸ (link) with slight modifications. Briefly, goat-anti human IgG F(ab’)2 (Invitrogen) was covalently coupled to yellow-green carboxylate beads (Thermofisher). Antibodies were diluted in culture medium to a starting concentration of 133 nM and serially diluted 4-fold 7 times. Diluted mAbs were incubated with anti-human IgG beads for 2 h at 37°C to form immune complexes. THP-1 (ATCC) cells (25,000/well) were added to the immune complexes and incubated at 37°C for 4 h. Cells were washed 2x with cold 1x PBS prior to being fixed with 4% paraformaldehyde. The cells were analyzed on a NovoCyte Advanteon flow cytometer (Agilent) (Figure S2C). A phagocytosis score was calculated as the (percentage of FITC+ cells) x (the geometric mean fluorescence intensity (gMFI) of the FITC+ cells)/100,000. Buffer only wells were used as negative controls and the assay was performed in technical replicate with two biological replicates.