Fluosphere neutravidin beads

For the ADNP assays^{40 (link)}, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (ThermoFisher). Immune complexes were formed by incubating antigen coupled beads with 10 ul of 1:50 diluted plasma samples at 37 °C for 2 hours. Primary neutrophils were isolated as previously mentioned and 50,000 cells per well incubated with washed immune complexes at 37 °C for 1 hour. Following incubation, neutrophils were stained for surface CD66b (Biolegend, 1:100, clone: G10F5) expression and fixed with 4% para-formaldehyde. Analysis was done on an iQue analyzer (Intellicyt).

For the ADCP assays^{41 (link),42 (link)}, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (ThermoFisher). Immune complexes were formed by incubating 10 ul of antigen coupled beads with 10 ul of 1:100 diluted plasma samples at 37 °C for 2 hours. THP-1 monocytes (25,000 cells per well) were then added to the beads then incubation at 37 °C for 16 hours. Following incubation, cells were fixed with 4% para-formaldehyde and analyzed on an iQue analyzer (Intellicyt).

THP-1 phagocytosis assay was performed as described before (50). In brief, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (Thermo Fisher Scientific) and incubated with 10 μl 1:100 diluted plasma for 2 hours at 37°C to form immune complexes. THP-1 monocytes (American Type Culture Collection) were added to the beads, incubated for 16 hours at 37°C, and fixed with 4% paraformaldehyde. Samples were analyzed on a iQue analyzer (Intellicyt). Each sample was analyzed in duplicate.

Phagocytosis score of primary human neutrophils was determined as described before (49). Biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (Thermo Fisher Scientific) and incubated with 10 μl 1:100 diluted plasma for 2 hours at 37°C to form immune complexes. Primary neutrophils were derived from Ammonium-Chloride-Potassium (ACK) buffer lysed whole blood from healthy donors (see above) and incubated with washed immune complexes for 1 hour at 37°C. Afterwards, neutrophils were stained for surface CD66b (BioLegend Cat# 305111, RRID:AB_2563293, Pacific Blue conjugated) expression, fixed with 4% paraformaldehyde and analyzed on a iQue analyzer (Intellicyt). Each sample was analyzed in duplicate using neutrophils from two different blood donors (biological duplicate).

For the ADCD assays^{39 (link)}, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (ThermoFisher). Immune complexes were formed by incubating antigen coupled beads with 10 ul of 1:10 diluted plasma samples at 37 °C for 2 hours. Following incubation, non-specific antibodies were washed off followed by incubation of the immune complex with guinea pig complement in GVB + + buffer (Boston BioProducts) at 37 °C for 20 min. To stop the complement reaction, 15 mM EDTA (Corning) in PBS was added to the immune complex. C3 deposited on beads were stained with anti-guinea pig C3-FITC antibody (MP Biomedicals, 1:100, polyclonal) and analyzed on an iQue analyzer (Intellicyt).

Complement deposition was performed as described before (48). In brief, biotinylated antigens were coupled to FluoSphere NeutrAvidin beads (Thermo Fisher Scientific) and, to form immune complexes, incubated with 10 μl 1:10 diluted plasma samples for 2 hours at 37°C. After non-specific antibodies were washed away, immune-complexes were incubated with guinea pig complement in GVB++ buffer (Boston BioProducts) for 20 min at 37°C. EDTA-containing phosphate-buffered saline (15mM) was used to stop the complement reaction and deposited C3 on beads was stained with anti-guinea pig C3-fluroescein isothiocyanate (FITC) antibody (MP Bio Cat# 0855385, RRID:AB_2334913, 1:100) and analyzed on an iQue analyzer (Intellicyt). Each sample was analyzed in duplicate.