Il 10 secretion assay

Phenotypic analysis of human B cell subsets was performed with the following antibodies: anti-CD19-PE-Cy7 (Beckman Coulter, Marseille, France), anti-CD27-BV421 (BD Biosciences, Heidelberg, Germany), anti-IgM-PerCP/Cy5.5 (BioLegend, CA, USA), anti-IgM-BV605 (BioLegend), anti-CD38-PE (BD Biosciences), anti-TNFR1(CD120a)-FITC (Miltenyi Biotech), anti-TNFR2(CD120b)-APC (R&D Systems, Inc., Minneapolis, MN, USA), and murine IgG_2A-APC (R&D Systems, Inc.) as isotype control where indicated. Cells were incubated in the dark for 30 min at 4°C in PBS with 0.5% FCS. Samples were acquired using a FACS LSRII SORP (BD Biosciences, Heidelberg, Germany), and cytometry data (LMD files) were analyzed with Kaluza software (Beckman Coulter). The aqua fluorescent reactive dye (LIVE/DEAD Fixable dead Cell stain Kit, Invitrogen, CA, USA) was used for definition of live and dead cells.
For staining of IL-10-producing B cells we used the IL-10 Secretion Assay (Miltenyi Biotech, Bergisch-Gladbach, Germany). B cells were stimulated for 40 h in culture medium with 1 µM CpG ODN 2006. Staining with anti-IL-10 was performed according to the protocol provided by the manufacturer with a prolonged incubation of cells labeled with IL-10 catch reagent (6 h) in presence of 0.25 µM CpG for restimulation. Cells were subsequently stained for expression of other surface markers before measurement on a flow cytometer.