Anti guinea pig c3 fitc antibody

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).

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).