Complement deposition of F. alocis by flow cytometry

F. alocis ATCC 35896 from 7-day old agar plate culture was harvested, washed once in PBS, adjusted to OD₆₀₀ of 0.5 and incubated for 45 min with 20 μM CFSE (Carboxyfluorescein succinimidyl ester). After pre-staining with CFSE, bacteria were washed once in GVB⁺⁺ (CP/LP) or Mg-EGTA (2.5 mM veronal buffer (pH 7.3) containing 70 mM NaCl, 140 mM glucose, 0.1% gelatin, 7 mM MgCl₂, and 10 mM EGTA; AP) and adjusted to OD₆₀₀ of 0.3. Thereafter, 140 μl bacteria were mixed with 1 – 30% NHS or Δ NHS diluted in GVB⁺⁺ or Mg-EGTA and incubated for 1 h at 37°C in a total volume of 200 μl. Similar experiments were performed using commercial NHS and deficient sera (Quidel). F. alocis ATCC 35896, treated as above, was incubated with 15% C1q-, C2-, factor B (FB)-deficient sera or NHS (all sera in active and heat-inactivated form) in GVB⁺⁺ or Mg-EGTA. Then bacteria were washed once in FACS buffer (50 mM HEPES, 100 mM NaCl, pH 7.4, 1% BSA, 30 mM NaN₃) and incubated with specific polyclonal antibodies (pAbs) against C3c (Dako) for 30 min at RT, followed by 30 min incubation with secondary F(ab′)₂ fragments conjugated with Alexa fluor 647 (Life Technologies). Using the same protocol C3b deposition on FITC-labelled zymosan (Molecular Probes) from selected sera was tested. To evaluate binding of different C3 protein fragments by F. alocis and M. catarrhalis, similar protocol was used, whereby bacteria were incubated with purified C3, C3b, C3met, C3d and C3c proteins in Binding Buffer (10 mM Hepes, pH 7.2, 1 mM MgCl₂, 2 mM CaCl₂) supplemented with 0.5% BSA and detection was performed with F(ab′)₂ fragments of the rabbit anti-C3d antibody (Dako) followed by the goat anti-rabbit Alexa-647 labeled secondary antibodies (Invitrogen). The sensitivity of the interaction to ionic strength was tested by the incubation of bacteria with C3met in the presence of increasing concentrations of NaCl starting from physiological amount (0.15–2.5 M NaCl). For testing effect of FACIN on deposition of C3b, NHS (5%) was supplemented with 3–100 μg/mL purified recombinant protein and the incubation with bacteria was carried out for 45 min. Finally, bacteria were washed twice, resuspended in fixing buffer (BD) diluted 1:10 in H₂O and analyzed using flow cytometry (CyFlow space, Partec, Germany). For M. catarrhalis, the C3b deposition was evaluated by the same procedure, except for using primary F(ab′)₂ fragments anti-human C3c during detection, due to problems with unspecific binding of pAbs by these bacteria. The geometric mean fluorescence intensity was calculated for all the samples using FlowJo software (Tree Star).