Vacutainer venous blood collection tubes sst

To isolate human neutrophils, peripheral blood was drawn into tubes containing trisodium citrate, citric acid, and dextrose (Vacutainer ACD Solution A, BD). Autologous serum, used for culture of cells was obtained by drawing blood into BD Vacutainer™ Venous Blood Collection Tubes SST, followed by centrifugation at 2500 × g for 30 min and serum collection. All blood donors provided written informed consent. For normal human blood samples, 10mls human blood was supplemented with GM-CSF (10 ng/ml) for 30 min at 37 °C followed by addition of 30 μg FITC-IgG isotype control or FcγRIIIB (3G8)-FITC-Ova conjugate for 2 h at the indicated concentrations. Blood was then incubated with Hetasep (STEMCELL Technologies) according to manufacturer protocols to deplete red blood cells and enrich leukocytes. Neutrophils were isolated from the leukocyte-rich plasma layer using a Easysep Neutrophil enrichment kit (STEMCELL Technologies). Neutrophil purity was evaluated using CD15, CD11b, CD66b, and lineage markers (CD3, CD19, and CD56) Supplementary Tables 4 and 6.

Peripheral blood was drawn into BD Vacutainer™ Venous Blood Collection Tubes SST. Tubes were centrifugated at 2500 × g for 30 min. Serum was aliquoted and frozen at −80 °C. To purify IgG, serum samples were incubated with Protein G high-capacity agarose beads (Genesee Scientific, #20–538). Antibody was eluted with 0.1 M Glycine (pH 2.7) and the samples were neutralized immediately with the neutralization buffer (1 M Tris-HCl, pH 9). The samples were then dialyzed against PBS overnight and concentrated using 100 kDa centrifugal filters (Millipore, UFC910024). To generate SLE-IgG/RNP immune complexes, solutions containing 2 mg/ml of purified SLE IgG or normal human IgG were mixed with equal volumes of 0.3 mg/ml of Sm/RNP complexes (#SRC-3000, Immunovision) in PBS (without Ca, Mg) and incubated for 4 h at 37 °C. 20 μl of immune complex solutions were added to 1 ml of neutrophil suspension. RNP was used at 3 μg/ml. SLE-IgG or normal human IgG alone were used at 20 μg/ml.