S monovette r hirudin tubes

Blood for serum collection was collected in SST II Advance tubes (BD, Ref. 367953). Tubes were inverted after blood was drawn, incubated for 15 minutes at room temperature to clot, centrifuged with 3000 × g for 15 min at room temperature and serum was stored in small aliquots at −80 °C.
Blood for plasma preparation was collected in K2E (EDTA) tubes (BD ref. 367864), Trisodium citrate tubes (BD ref. 363047), Sodium heparin tubes (BD ref. 367869), Lithium heparin tubes (BD Ref. 368496) or S-Monovette r-Hirudin tubes (Sarstedt, ref. 04.1944.001). Tubes were inverted after blood was drawn, centrifuged with 3000 × g for 15 min at 4 °C and plasma was stored in small aliquots at −80 °C.
For human IgG, human IgM and human C3 binding, bacteria (1.10E7 in 100 μL) were incubated with 10% plasma or serum in Hank’s Balanced Salt Solution (HBSS) without phenol red containing Ca²⁺/Mg²⁺⁺ 0.1% gelatin (HBSS3+) for 30 min at 37 °C. Bacteria were washed and incubated with 1:500 diluted FITC-labelled poly-clonal goat anti-human C3 (MP biomedicals), 1:100 diluted FITC-labelled Fc-specific goat anti-human IgG (Sigma-Aldrich) or 1:100 diluted FITC-labelled μ-chain-specific goat anti-human IgM (Sigma-Aldrich) in PBS with 2% BSA for 30 min at 4 °C. Bacteria were washed and fixed for 20 min with 2% paraformaldehyde. Bacteria were taken up in PBS for flow cytometry.

Cord blood or peripheral venous blood of healthy human donors was collected in S-Monovette r-Hirudin tubes (Sarstedt) to preserve complement activity (36 (link)). The blood was centrifuged at 1000x g for 5 minutes and plasma was removed and stored immediately at -80°C. Plasma of 27 healthy donors was pooled before freezing and cord blood plasma was stored individually. For complement inactivation, the plasma was heat-inactivated for 30 minutes at 56°C.