Non reactive isotype matched control monoclonal antibodies

Splenocytes were isolated using a 40-µm nylon cell strainer (BD Biosciences, San Jose, CA, USA); RBCs were lysed with buffer containing 0.14 NH₄Cl and 0.017 M Tris-base (pH 7.5). IL-10-producing CD1d^highCD5⁺CD19⁺ Bregs were analyzed by flow cytometry after immunostaining of surface markers with the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD19 (clone 1D3) (BD Biosciences, San Jose, CA, USA), phycoerythrin (PE)-conjugated anti-CD1d (clone 1B1; isotype, Rat IgG2b, κ) (BD Biosciences), allophycocyanin (APC)-conjugated anti-CD5 (clone 53–7.3) (eBioscience, San Diego, CA, USA), and peridinin chlorophyll-protein complex (PerCP)- or PE-conjugated anti-IL-10 (clone JES5–16E3) (eBioscience). For subsequent intracellular IL-10 staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's protocol (BD Biosciences). Intracellular transport processes were inhibited with BD GolgiStop containing the transport inhibitor monesin and Fc receptors were blocked by treatment with rat anti-mouse CD16/32 antibody (BD Bioscience) for 15 min. The FlowJo software (Tree Star Inc., Ashland, OR, USA)was used to analyze the flow cytometry data. To determine background staining, non-reactive isotype-matched control monoclonal antibodies (eBioscience) were used and cells were gated to exclude ≥98% of non-reactive cells.

For flow cytometry analysis of IL-10-producing CD19⁺ B_regs, cells in splenocytes were analyzed for the expression of surface markers using the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD19 (clone 1D3; BD Biosciences, San Jose, CA, USA) and phycoerythrin (PE)-conjugated anti-IL-10 (clone JES5-16E3; eBioscience, San Diego, CA, USA). For subsequent IL-10intracellular staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's protocol (BD Biosciences). For the analysis of CD4⁺CD25⁺FoxP3⁺ T_reg cells, the mouse T_reg-staining kit was used according to the manufacturer's instructions (eBioscience). The stained cells were analyzed by 3-5 color flow cytometry using a FACSversa flow cytometer (BD Biosciences). The FlowJo software (Tree Star Inc., OR, USA) was used to analyze flow cytometry data. To determine background staining, non-reactive iso-type-matched control monoclonal antibodies (eBioscience) were used and gated to exclude ≥98% of non-reactive cells.