Gvb buffer

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.

Complement deposition was performed as previously described [14 (link)]. Briefly, biotinylated antigens were bound to FluoSphere NeutrAvidin beads (Thermo Fisher Scientific; Waltham, MA, USA). To form immune-complexes, antigen-coated beads were incubated with 10 µL of 1:10 diluted serum samples. Non-specific antibodies were washed away, and immune complexes were incubated with guinea pig complement in GVB++ buffer (Boston BioProducts; Milford, MA, USA). Complement reaction was stopped using EDTA containing PBS (15 mM). Deposited C3 on beads were stained with anti-guinea pig C3-FITC antibody (MP Biomedicals; Irvine, California USA, 1:100, polyclonal) and analyzed on an anti-guinea pig C3-FITC antibody (MP Biomedicals, CA, USA, 1:100, polyclonal) and analyzed on an iQue analyzer (Intellicyt; Albuquerque, NM, USA).

Complement deposition was performed as described previously^{49 (link)}, using Luminex beads (Luminex Corp). Briefly, Luminex microspheres were coupled to biotinylated antigens, and immune complexes were formed using 1:30 diluted plasma samples. After a 2-h incubation at 37 °C, guinea pig complement in GVB++ buffer (Boston BioProducts) was added to immune complexes for 20 min at 37 °C. To stop the complement reaction, EDTA-containing phosphate-buffered saline (15 mM) was used, then C3 deposition on beads was detected using a 1:100 diluted anti-guinea pig complement C3 antibody (MP Biomedicals). MFI values were analyzed by flow cytometry on an iQue analyzer (Intellicyt) (Fig. S6).