Streptococcal Complement Deposition Assay

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)].