Cells were incubated with the antibodies for a minimum of 30 min at 4 °C in darkness followed by two washes with PBS. Pelleted cells were resuspended in PBS containing 5% FCS (Sigma-Aldrich, St Louise, MO, USA) at the concentration of 10 × 106 cells/ml and using BD Bioscience FACSAria sorted with respect to their CD19 and CD25 expression rendering two highly purified (>98.5%) B-cell populations, CD19+ CD25+ and CD19+ CD25−. Gating strategy for sorting is shown in Fig. 1. Supernatant preparation for cytokine measurements. CD19+ CD25+ or CD19+ CD25− B cells were plated at a concentration of 2.5 × 105/ml
in a volume of 100 μl per well in round-bottom 96-well plates (TPP, Switzerland). selleck products Iscove’s medium containing, 10% FCS, 1% gentamicin, 1%l-glutamine
and 1% mercaptoetanol (all from Sigma-Aldrich, and hereafter called complete Iscove’s medium) was used. The different B-cell populations were stimulated with either 3 μm backbone protected CpG-PS (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′, Scandinavian Gene Synthesis AB, Köping, Sweden), 5 μg/ml E-coli LPS (Sigma-Aldrich, St Louis, MO, USA) or 0.5 μg/ml Pam3Cys (EMC Microcollections, Tübingen, Germany) in a humidified atmosphere containing 5% CO2 at 37° for 12, 48 and 72 h. Neat cells incubated at the same conditions were used as controls. Supernatants collected were stored this website at −70° until used. IL-6 bioassay. To measure IL-6 levels in the supernatants, we used a cell line B13.29 (B9 cells)
which depend on IL-6 for its growth . In flat-bottom 96-well plates, 5 × 103 B9 cells per well were incubated (TPP, Switzerland) in complete Iscove’s medium. Supernatants were diluted 1:25 or 1:250 and added in triplicates to the B9 cells. Recombinant mouse IL-6 (National Institute of Biological Standards PAK6 and Control, Hertfordshire, UK) was used as a standard. After 72 h of culture, cells were pulsed with 1 μCi 3H-thymidine (Amersham Pharmacia Biotech) and harvested after 6 h on glass fibre filter (Walluc Oy, Turku, Finland). The incorporated 3H-thymidine was measured using a β-scintillation counter. Cytometric Bead Array. To measure the release of IL-2, IL-4, interferon-gamma (IFN-γ) and tumour necrosis factor (TNF), we used Cytometric Bead Array flex set (BD Bioscience) according to the manufactures protocol. Analyses were made on the supernatants from 24 to 72 h. IL-10 ELISA. Mouse IL-10 ELISA was purchased from R&D systems (Abingdon, UK) and performed according to the manufacturer’s recommendations. The optical density of the samples was determined using wavelengths 540–570 nm on a SpectraMax Plus (Molecular Devices, Sunnyvale, CA, USA). Mixed lymphocyte reaction (MLR). Spleen cells from C57BL/6 mice were sorted as described previously and irradiated with 2500 rad.