After stimulation with cytokines, B cells were washed with phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS, endotoxin-free; Cambrex,

Verviers, Belgium) and their phenotype was analysed by flow cytometry as described above. Cell-free supernatants were stored at −20°C until utilized. Using naive CD27- B cells, we measured the level of Ig produced after CSR. In our experiments, the majority (90·5 ± 4·6%) of freshly isolated B cells were naive IgD+IgM+ B cells. In certain experiments, B cells were cultured for 120 min in supplemented Iscove’s modified Dulbecco medium (IMDM). Blocking RAD001 chemical structure antibodies (5 µg/ml) against IL-6R, IL-10Rα and/or IL-10Rβ (clones 17506, 37607 and 90220, respectively; R&D Systems, Lille, France) were added with sCD40L and cytokines at the start of B cell culturing and monitored for 12 days. Binding of the IL-6R blocking antibodies on B cells was assessed by flow cytometry daily throughout the culture period (12 days, data not shown) [25]. IL-6 (48 h) and Ig total (12 days) levels in cell-free supernatants were quantified using a commercial specific enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems), according to the manufacturer’s

instructions [14,23,24]. ELISA plates (BD Biosciences) were coated with F(ab′)2 of goat IgG anti-human IgA, IgG or IgM (33 ng/ml; MP Biomedical, Illkirch, France). After an overnight incubation at 4°C and four washes, plates were blocked for 60 min with PBS containing 1% bovine serum albumin (BSA). Supernatants at a 1:10 dilution were applied to the samples and incubated

for 60 min at 37°C. After incubating for 45 min at 37°C, the plates were washed and bound Ig was detected Carfilzomib in vivo with a horseradish-peroxidase (HRP)-labelled goat F(ab′)2 IgG of anti-human Protein tyrosine phosphatase IgA, IgG or IgM (Sigma-Aldrich). After four washes, O-phenylendiamine dihydrochloride (Sigma-Aldrich) was added and the plates were incubated at room temperature in the dark for 20 min. The reaction was stopped by addition of 1 M HCl (Sigma-Aldrich). Purified B cells were incubated for 30 min, as described previously [26], with 50 ng/ml of sCD40L and 100 ng/ml of IL-10, with or without 5 ng/ml of IL-6. The cells were then washed with PBS–FBS (Cambrex) and treated with a nuclear extraction kit (Active Motif, Rixenart, Belgium), according to the manufacturer’s instructions. Cytoplasmic and nuclear extracts were obtained for each condition and were stored at −80°C until used. The levels of phosphorylated NF-κB p65 (pNF-κB p65, assay sensitivity = 0·5 µg/well) and phosphorylated STAT3 (pSTAT3, assay sensitivity = 0·6 µg/well) in the nuclear extracts of stimulated and non-stimulated B cells from each cell culture condition was determined using a transcription factor ELISA kit (active motif). Briefly, 2·5 µg of each nuclear extract was incubated in 96-well plates coated with a consensus sequence nucleotide binding site for pNF-κB p65 (5′-GGGACTTTCC-3′) or for pSTAT3 (5′-TTCCCGGAA-3′).