All samples including standards were determined in duplicate. Sample values were calculated from the curve fitted to the readings of the standard (using Ascent software v. 2.6, Thermo Scientific). The detection limit of the assay was 0. 5 μg/ml. Immunocytostaining and Flow Cytometry Single-cell suspensions were prepared from spleens and transferred to round-bottomed 96-well polystyrene plates (NUNC, Roskilde, Denmark) with 3 × 105 cells/well. Fcγ III/II (3 μg/ml, 50 μg/ml; BD Biosciences) was added for 10 minutes to block non-specific binding of antibodies. An additional 50 μl/well PBS-Az containing fluorochrome-conjugated check details antibodies

at various concentrations was added and the cells were incubated for 45 minutes. The cells were then washed and resuspended in 200 μl/well PBS-Az containing 2% formaldehyde for flow cytometric analyses. All stainings were carried out at or below 4°C. The antibodies used in this study were APC-conjugated Enzalutamide molecular weight anti-mouse CD4, clone RM4-5 (rat IgG2a,κ); PE-conjugated anti-mouse CD3e, clone 145-2C11 (Armenian hamster IgG); APC-conjugated anti-mouse CD8a (Ly2), clone 53-6.7 (rat IgG2a, κ); APC-conjugated anti-mouse CD49b, clone DX5 (rat IgM, κ); PE-conjugated anti-mouse CD19, clone 1D3 (rat IgG2a, κ); APC-conjugated anti-mouse CD11c, clone N418 (Armenian hamster IgG);

APC-conjugated anti-mouse Ly-6G (Gr-1), clone RB6-8C5 (rat IgG2b, κ) and isotype controls for rat IgG2a, κ; rat IgG2b, κ; Armenian hamster IgG1, clone eBio299Arm; rat IgM, κ, all purchased from eBioscience. diglyceride Stained cells were analysed

on a BD FACSArray flow cytometer (BD Biosciences) and data was analysed using FCS Express 3.0 software (De Novo Software, CA). In vitro fermentation of non-digestible dietary carbohydrates The fermentation study was performed using a basal medium containing: peptone water (2 g/L, Oxoid), yeast extract (2 g/L, Oxoid), NaCl (0.1 g/L, Merck), K2HPO4 (0.04 g/L, Merck), KH2PO4 (0.04 g/L, Merck), MgSO4·7H2O (0.01 g/L, Merck), CaCl2·6H2O (0.01 g/L, Sigma-Aldrich), NaHCO3 (2 g/L, Merck), haemin (0.005 g/L, Sigma-Aldrich), L-cystein HCL (0.5 g/L, Sigma-Aldrich), bile salts (0.5 g/L, Oxoid), Tween 80 (2 ml/L, Merck), vitamin K1 (10 μl/L, Sigma-Aldrich), resazurin (0.001 g/L, Sigma-Aldrich) and 1% (wt/vol) test carbohydrate (inulin, FOS, XOS, GOS, beta-glucan, apple pectin, polydextrose and glucose) [42]. Stock solutions of peptone water, NaCl, K2HPO4, KH2PO4, CaCl2·6H2O, MgSO4·7H2O and NaHCO3 were prepared and autoclaved (121°C, 15 min.). Appropriate volumes of the stock solutions were mixed, autoclaved and supplemented with Anlotinib datasheet sterile filtered (0.2 μm) solutions of bile salts, L-cystein HCL, resazurin and yeast extract. Furthermore, haemin, Tween 80 and vitamin K1 were added.