Blood lymphocytes were washed once in cold PBS, and following centrifugation (680 g, 18°C, 10 min) the pellet was resuspended in 2 ml red blood cell lysis buffer [0·15 m NH4Cl, 0·01 m KHCO3 and 10 µm ethylenediamine tetraacetic acid (EDTA) Na2·2H2O] and incubated for 2 min at room temperature. The volume was then adjusted to 30 ml using

sterile PBS and centrifuged. Following two subsequent washes, the cell pellet was resuspended in IMDM (Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco, Mulgrave, Australia) and anti-mycotic solution (10 mg/l; Sigma). PBMCs/CRL-9850 cells were plated in six-well tissue culture plates (Corning, Sigma) at 5 × 106 cells/well and incubated at 5% CO2, 37°C for 24 h prior to stimulation with bacteria, as described by Amrouche et al. [21]. Briefly, 106 freshly prepared viable (live or GIT) or equivalent (∼106 CFU/ml) heat-killed bacteria were added per 106 cells Cobimetinib molecular weight and co-cultured for 72 h at 5% CO2, 37°C. At 6, 12, CP-673451 chemical structure 24, 48 and 72 h, 500 µl samples of the culture medium were collected and analysed for cytokine secretion by

ELISA (Becton Dickinson, San Jose, CA, USA), in accordance with the manufacturer’s instructions. Data are expressed as the mean cytokine response minus background (pg/ml) of each treatment from triplicate wells, plus or minus the standard error of the mean. Treg/Th17 populations were characterized following PBMC/bacteria co-culture. Briefly,

106 PBMC were co-cultured with either live or killed bacteria, lipopolysaccharides (LPS; Sigma) or media alone, in a 24-well plate at 37°C in 5% CO2 for 96 h, then cells were washed twice using FACS buffer (PBS + 2% FCS) and centrifuged at 500 g for 10 min. PBMC were resuspended at 106 cells/ml, and surface marker staining was performed using fluorescein isothiocynate (FITC)-labelled anti-human CD4, allophycocyanin-labelled anti-human CD25/CD3 (Becton-Dickinson), peridinin chlorophyll protein (PerCP)-labelled anti-human CD3 (Biolegend, San Diego, CA, USA) and PerCP cyanine (Cy)5·5-labelled anti-human CCR6 (CD196). Intracellular staining was performed using phycoerythrin (PE)-labelled anti-human FoxP3/RORγt (BD MG-132 ic50 Pharmingen and R&D Systems, Minneapolis, MN, USA, respectively), according to the manufacturer’s instructions. Samples were read using a BD FACSCalibur, data acquired using CellQuest program (Becton Dickinson Biosciences), and analysis performed using Gatelogic version 3·07 software (Inivai, Victoria, Australia). Absolute numbers of Treg cells and Th17 cells were calculated as a percentage of the total lymphocyte number. All co-cultures were carried out in triplicate. Results obtained were analysed as a split plot in time design with three main factors: strains (six levels) and treatments (three levels) as the main plot and time (five levels) as a subplot.