ADNP Assay for Neutrophil Phagocytosis

The ADNP assay was adapted from Karsten et al. (56 (link)). Antigens were coupled to beads and immune complexes were formed as described for ADCP. Neutrophils were isolated from freshly drawn whole blood. Erythrocytes were lysed with ammonium-chloride potassium lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂ EDTA, pH 7.4), and leukocytes were separated out by centrifugation, 500g for 5 minutes at room temperature. Leukocytes were washed with cold PBS, resuspended in R10, and added to plates at a concentration of 5 × 10⁴ cells/well. The plates were incubated for 1 hour at 37°C. The neutrophil marker CD66b (Pacific Blue–conjugated anti-CD66b; BioLegend, 305112) was used to stain cells. Cells were fixed for 20 minutes in 4% paraformaldehyde (PFA). Fluorescence was acquired with an Intellicyt iQue, and the phagocytic score was calculated as described for ADCP.