Fig 1: Interaction between complement system and peptidorhamnomannans (PRMs) of Sporothrix species. (A) Sporothrix yeasts were opsonized with C3b and labeled with mouse anti-C3b mAb and mouse TRITC-IgG (Ab). PRMs (50 µg/ml in 50 mM carbonate buffer, pH 9.6) were coated on 96-well plate: their (B, C) C3 activation capacities were determined using GVB+ or GVB- (with EGTA) media using whole/complement factor-depleted human sera. (D) Interaction with CD11b was determined by sequential addition of CD11b, mouse anti-CD11b mAb, peroxidase-conjugated mouse IgG, and chromogenic substrate for peroxidase (tetramethyl benzidine); colors developed were optically read at 450 nm. For each condition, two independent experiments, in triplicate, were performed. Statistical analysis was performed by ANOVA with Tukey’s multiple comparison posttest (****p < 0.0001).
Fig 2: (A) Cytokine released by human monocyte-derived macrophages (hMDMs) blocked for complement receptor-3 (CR3) and challenged with Sporothrix species. Sporothrix yeasts were made to interact with hMDMs blocked for CR3 (with monoclonal anti-CD11b antibodies) or unblocked (infected control) in a culture medium supplemented with whole human serum (wHS) for 18 h at 37°C and 5% CO2. Non-infected controls were hMDMs blocked with irrelevant antibodies and treated with culture medium supplemented with wHS. The culture supernatants were collected and analyzed for tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Results indicate the mean ± SEM of at least two independent experiments in duplicate or triplicate (n = 4 or 6). ANOVA with Tukey’s multiple comparison posttest was performed for the statistical analysis (***p < 0.001, **p < 0.001). (B) Uptake of Sporothrix yeasts by hMDMs after CR3 blockage. hMDMs were incubated with monoclonal anti-CD11b antibodies for 1 h and challenged with FITC-labeled yeasts of Sporothrix schenckii or Sporothrix brasiliensis. After 4 h of interaction in the presence of medium containing wHS, hMDMs were analyzed by FACS to determine the change in mean binding index (MBI) compared to control (=CD11b unblocked hMDMs interacted with FITC-labeled Sporothrix yeasts). Results are the mean ± SD of an experiment performed in duplicate.
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