Veronal buffer

Plasma was prepared by centrifugation of fresh whole blood at 2500 Xg for 10 minutes. Equal volumes of plasma, Veronal buffer (Boston Bioproducts, MA, USA) and the samples were mixed together and incubated at 37°C for 30 min. The samples were stored in −80°C before use. The amount of iC3b was detected by Quidel iC3b ELISA Immunoassay (EIA) MicroVue Complement kit (Thermo-Scientific, Pittsburgh, PA) following manufacturer’s protocol. Cremophor (EMD Millipore, MA, USA) and 0.9% saline solution were used as positive and negative controls, respectively.

Human blood containing sodium citrate as the anti-coagulant was subjected to centrifugation at 2500g for 10 minutes and plasma was collected. In a microcentrifuge tube, 100 μL each of Veronal buffer (Boston Bioproducts, Ashland, MA), plasma and test sample were mixed and incubated at 37 °C for 30 minutes. Appearance of iC3b was measured using the MicroVue iC3b EIA kit (Quidel Corp., San Diego, CA) following manufacturer’s instructions. As a positive control, the vehicular composition of Taxol® was prepared by mixing Cremophor-EL (Milliore Sigma, Burlington, MA) 1:1 with ethanol containing 2 mg/mL citric acid.

GP-coated beads were generated as described for ADNP, but with
substitution of red-fluorescent Neutravidin beads (Life Technologies).
Antibodies were diluted in culture medium to 5μg/ml and incubated
with GP-coated beads for 2 h at 37 °C. Unbound antibodies were
removed by centrifugation prior to the addition of reconstituted guinea pig
complement (Cedarlane Labs) diluted in veronal buffer supplemented with
calcium and magnesium (Boston Bioproducts) for 20 min at 37 °C. Beads
were washed with PBS containing 15 mM EDTA, and stained with an
FITC-conjugated anti-guinea pig C3 antibody (MP Biomedicals). C3 deposition
onto beads was analyzed by flow cytometry. The gMFI of FITC of all beads was
measured. Gating strategy and representative flow cytometry plots are shown
in Figure S4.