Fitc labelled polyclonal goat anti human c3

Bacteria were grown in Tryptic Soy Broth to OD₆₂₀ ~ 0.23, washed once with HBSS + Ca²⁺/Mg²⁺ + 0.1% (w/v) gelatin (HBSS3+) and diluted to an OD₆₂₀ of 0.2 with HBSS3+. Twenty‐five microlitre bacteria was mixed with 25 μL 20% (v/v) patient serum diluted in HBSS3+ with or without reconstitution of 0.2 μg mL⁻¹ FD (CompTech) and/or 30 μg mL⁻¹ anti‐C1q and incubated 30 min at 37°C. Bacteria were pelleted by centrifugation at 3200× g and fixed for 20 min in 2% (w/v) paraformaldehyde in PBS at RT. Surface‐bound complement C3 and complement complex C5b‐9 were detected with 1:500‐diluted FITC‐labelled polyclonal goat anti‐human C3 (MP biomedicals) and 1:100‐diluted monoclonal mouse antihuman C5b‐9 (Clone aE11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by 1:500‐diluted Alexa647‐labelled donkey antimouse IgG (Invitrogen). Surface binding of C3 and C5b‐9 was determined by flow cytometry using a FACS LSR II instrument (BD Biosciences, San Jose, CA, USA) and expressed in mean fluorescence intensity (MFI) in arbitrary units (AU). Data were analysed by using FlowJo version 10.4.1 (BD Biosciences).

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.

Frozen bacteria stocks were thawed, washed once with HBSS + Ca²⁺/Mg²⁺ + 0.1% gelatin (HBSS3+) and diluted to an OD₆₂₀ of 0.1 with HBSS3+. A volume of 25 µL of bacteria were mixed with 25 µL 10% human serum (for C3 binding) or 10% heat‐inactivated (HI) (20 min 56°C) serum (for IgG binding) in HBSS3+ with or without reconstitution of fD (A136; CompTech) reconstitution and incubated 0, 5, 15 or 30 min at 37°C. Complement activation was stopped by adding 5 mm EDTA and cooling on ice. Bacteria were pelleted by centrifugation at 3200 g and fixed for 20 min in 2% paraformaldehyde in PBS at room temperature. Surface‐bound complement C3 was detected with 1:500‐diluted FITC‐labelled polyclonal goat anti‐human C3 (MP Biomedicals, Solon OH, USA). Surface‐bound IgG was detected with 1:500‐diluted Fcγ fragment‐specific PE‐labelled AffiniPure goat anti‐human IgG (Jackson ImmunoResearch, West Grove, PA, USA). Surface binding of C3 or IgG was determined by flow cytometry using a FACS LSR II instrument (BD Biosciences) and expressed in mean fluorescence intensity in arbitrary units. Data were analysed by using FlowJo version 10.4.1. (FlowJo LLC, Ashland, OR).