The procedure was performed

according to the instructions of the manufacturer and the acquisition and analysis was performed as described previously (Vissers et al., 2010). Proliferation was studied by intracellular expression of the nuclear Ki-67 antigen (BD Pharmingen, San Diego, CA) by flow cytometric analysis. Cultured cells were collected on both 4 and 8 days of culture. In each assay, 5 × 105 hPBMC were incubated with 100 μL cytofix/cytoperm (BD Pharmingen) for 15–20 min on ice to fix and permeabilize the cells. Cells were washed twice with perm/wash buffer (BD Pharmingen) and incubated with Selleckchem BGB324 an anti-Ki-67 PE antibody (or the matched isotype control) diluted in perm/wash buffer for 30 min on ice in the dark. Hereafter, the cells were washed once again with the perm/wash buffer, resuspended in PBS and measured selleck chemicals llc on the flow cytometer. Values are expressed as the percentage of stimulated cells positive for the Ki-67 mAb corrected for the percentage of stimulated cells that were positively stained by the isotype control. Cytokine production by hPBMC was analyzed in supernatants of cells cultured for 1, 4 and 8 days. The production of the innate and

adaptive cytokines IL-1β, IL-10, IL-12p70, IL-13, IFN-γ and TNF-α was detected using cytometric bead array (cba; BD Biosciences). All buffers used in this protocol were obtained from the BD CBA Soluble Protein Master Buffer Kit (BD Pharmingen) and the procedure was performed according to the manufacturer’s protocol. The detection limits according to the manufacturer were as follows: 1.1 pg mL−1 IL-1β, 2.3 pg mL−1 IL-10, 2.2 pg mL−1 IL-12p70, 1.6 pg mL−1 IL-13, 0.3 pg mL−1 IFN-γ and 0.7 pg mL−1 TNF-α. The samples were measured on the FACSCanto II, using fcap software (BD Biosciences). Because of a nonnormal distribution PLEKHB2 of most of the data the nonparametric Wilcoxon signed-rank test was used. This test allowed to compare data from cultures in the absence of a bacterial strain with cultures in the presence of the different

strains and to compare data from cultures of different strains. The Wilcoxon signed-rank test was also used to compare cytokine data on different days and to compare cytokine data on day 8 of not-restimulated and restimulated cells. When P<0.05, the difference was considered to be statistically significant. The statistical analysis was performed using spss software (version 15.0; SPSS Inc., Chicago). Experimental data are presented as mean ± SEM. Although differences in hPBMC subset composition were observed between the different donors, all values were within the normal range of leukocytes present in the peripheral blood as assessed by Erkeller-Yuksel et al. (1992) and Jentsch-Ullrich et al. (2005) (data not shown). Viability of hPBMC directly after isolation was above 80% for all donors and the percentage late apoptotic/necrotic cells was below 5% (data not shown).