Quantifying Neutrophil Phagocytosis Capacity

Phagocytosis score of primary human neutrophils was determined as described before.³⁰ (link) Antigens were biotinylated with NHS-Sulfo-LC-LC kit according to the manufacturer’s instruction (Thermo Fisher). Excessive biotin was removed by size exclusion chromatography using Zeba-Spin desalting columns (7 kDa cutoff, Thermo Fisher). Biotinylated antigens were coupled to fluorescent neutravidin beads (Thermo Fisher) and incubated with 1:10 diluted plasma. Primary cells were derived from Ammonium-Chloride-Potassium (ACK) buffer lysed whole blood from healthy donors and incubated with immune complexes for one hour at 37°C. For the Fc-receptor blocking experiments, isolated neutrophils were pre-incubated with 5 μg/ml of FcγR2a (CD32A, clone IV.3, Bio X Cell Cat# BE0224, RRID:AB_2687707) and FcγR3 (CD16, clone: LNK16, Bio-Rad, RRID:AB_324304) five minutes prior to addition of neutrophils to the immune complexes. Neutrophils were stained for surface CD66b (BioLegend Cat# 305112, RRID:AB_2563294) expression, fixed with 4% para-formaldehyde, and analyzed an iQue analyzer (IntelliCyt) (Figure S8).