Fitc labeled cross reactive rabbit anti human c3c antibody

Complement deposition on the streptococcal surface was assessed basically as described [10], with the differences of the use of an IgG depleted porcine anti-S. suis strain 10 serum as source of IgM and complement. Briefly, S. suis strain 10, 10∆ide_Ssuis, 10∆ide_Ssuis ∇ide_Ssuis_EcoRI, ∆ide_Ssuis∇ide_Ssuis_C195S were grown to an OD₆₀₀ of 0.8. Then, 75µl of the respective cultures were centrifuged and incubated for 1h at 37°C on a rotator with 150 µl of a 1:2 dilution of an anti-S. suis strain 10 hyperimmune serum depleted of all IgG. Staining of C3 labeled bacteria was conducted with 250µl of a 1:150 diluted FITC-labeled cross-reactive rabbit anti-human C3c antibody (Dako, catalogue #F020102-2) for 1h at 4°C under constant rotation. As positive control, undiluted serum was used, as negative control, serum that had been heat inactivated at 56°C for 30 min was used. For samples with the addition of recombinant protein, 5µg of rIde_Ssuis or rIde_Ssuis_C195S were added to 10∆ide_Ssuis prior to incubation with serum. Samples were measured using BD, FACSFortessa and analyzed using FlowJo^TM_V10 software.

Deposition of complement on the streptococcal surface was assessed using flow cytometry assays as described previously [23 (link), 31 (link)]. Briefly, C3 deposition was investigated by incubating 2 × 10⁶ CFU of S. suis in 50 μL of CDS for 30 min at 37 ℃ under rotation (8 rpm). As negative control CDS was incubated for 30 min at 56 ℃ to inactivate all complement factors. Staining of C3-labeled bacteria was conducted with 200 µL of a 1:150 diluted FITC-labeled cross-reactive rabbit anti-human C3c antibody (Dako, F020102-2, 3 g/L) for 1 h at 4 ℃. Samples were measured using BD FACS Fortessa and analyzed using FlowJo^TM_V10 software. Results of complement binding assay are presented a fluorescence index (FI; percentage of positive bacteria multiplied by the geometric mean fluorescence intensity) in arbitrary units [32 (link), 33 (link)].