One µg of the mRNA was reverse-transcribed into cDNA with a master mix of oligo-dT (20 µg/ml, Roche, Meylan, France), deoxyribonucleotide (dNTP) (16 µmol/ml;

Invitrogen), RNase block (20 U/ml; Stratagene, Amsterdam, selleck chemicals llc the Netherlands) and reverse transcriptase (50 U/ml; Invitrogen). The cDNA was then PCR-amplified with β-actin housekeeping gene-specific primers (R&D Systems) designed to amplify a portion of the coding sequences (7·5 pmol/µl), dNTP (8 µmol/ml) and Taq polymerase (1·25 U/ml; Sigma-Aldrich). Raji B cells were used as positive amplification controls and a master mix without added cDNA was used as a negative control. The cDNA expression was detected on a 1·5% agarose gel. The final product of the β-actin housekeeping gene was 298 base pairs (bp) in size. To analyse AID gene expression, a nested reverse transcription–polymerase chain reaction (RT–PCR) assay was used. We selected the conserved active site of cytidine see more deaminase as the primary target. Primers

were designed as follows: external 5′ GAAGAGGCGTGACAGTGCT 3′ (sense) and 5′ CGAAATGCGTCTCGT AAGT 3′ (anti-sense); internal 5′ CCTTTTCACTGGACTTTGG 3′ (sense) and 5′ TGATGGCTATTTGCACCCC 3′ (anti-sense). The final product of the AID gene was 656 bp in size [27]. Quantification of band intensity was carried out by Image J version 1·42q software (National Institutes of Health, Bethesda, MD, USA) and expressed as the mean of the optical density of five independent blots ± standard error

of the mean (s.e.m.). Band intensity was normalized to the optical density of the actin-β housekeeping control loaded onto the same blot. Interexperimental comparisons of the cell culture conditions were analysed by a Mann–Whitney unpaired test. Differences were considered statistically significant for P < 0·05. The peripheral blood of normal healthy donors (n = 15) showed large variation in the frequencies of the peripheral B cell subsets (Fig. 1c), with 68·3 ± 8·9% IgD+CD27-, 11·5 ± 5·2% IgD+CD27+ and 22·9 ± 7·8% IgD-CD27+ B cells. The IgD-CD27+ B cells population could be subdivided further into 13·1 ± 3·2% IgD-CD27+IgG+ or IgD-CD27+IgA+ and 9·8 ± 3·6% IgD-CD27+IgM+ B cells. The optimal concentration of activators in this culture Y-27632 solubility dmso system required a balance between the best readout (IgA synthesis determined by ELISA) and B cell pathway activation (determined by Western blot). In agreement with previously published culture conditions, we selected the concentrations of 50 ng/ml for sCD40L, 100 ng/ml for IL-10 and 0·2 ng/ml for TGF-β. Although sCD40L or IL-10 alone increased IgA production significantly by approximately 10-fold and approximately 30-fold, respectively, IgA production after the simultaneous addition of sCD40L and IL-10 was statistically similar to that observed with addition of IL-10 alone (Fig. 2a). An additive effect was observed for IgA production when sCD40L was used at 50 ng/ml and IL-10 from 80 to 120 ng/ml (Fig. 2b).