Hamster anti mouse cd28

SiLP and splenic cells were also seeded on 24-well plates at 1 × 10⁶ cells per well for cytokines assays in Cerrotini culture medium (Malaisé et al., 2018) in presence or absence of 5 μg/mL hamster anti-mouse CD3 and hamster anti-mouse CD28 (BD biosciences) coated wells. Culture supernatants were collected after 3 days of stimulation and frozen at − 80 °C prior to cytokines secretion measurement. Cytokines were also analyzed in supernatant of jejunal, colonic fragments or feces resuspended in RIPA buffer as previously described^{26 (link)}. Jejunal, colonic or fecal protein concentrations were measured using BCA uptima kit (Interchim).
IL-17, IFN-γ and lipocalin were assayed using commercial ELISA kits (R&D Systems, Lille, France), following manufacturer’s instructions.

CD4⁺ T cells were isolated from the mesenteric lymph nodes (MLN) using a commercial cell isolation kit and following the manufacturer's instructions (Miltenyi, Biotec Inc.; San Diego, CA). Cells from each animal were plated (10⁶ cells/well), cultured and polarized to Th17 cells in RPMI supplemented with 2-β Mercaptoethanol (50 μM), immobilized hamster anti-mouse CD3ε (500 μg/ml: BD biosciences), hamster anti-mouse CD28 (2 μg/ml: BD biosciences, San Diego, CA, USA), rat anti-mouse IFN-γ (10 μg/ml: BD biosciences, San Diego, CA, USA), rat anti-mouse IL-4 (10 μg/ml: BD biosciences, San Diego, CA, USA), hTGF-β (5 ng/ml: Peprotech, Inc, UK), recombinant murine IL-6 (20 ng/ml: Peprotech, Inc, UK) recombinant murine IL-23 (BioLegend, CA, USA, 5 ng/ml). Three days later the cells were stimulated for 5 h with PMA (100 ng/ml: Tocris Bioscience, Bristol, UK) and ionomycin (free acid, 500 ng/ml: Tocris Bioscience, Bristol, UK), and the supernatants were collected and stored at −20°C for cytokine measurement.

To culture, cells were seeded on 24-well plates at 1 × 10⁶ cells per well for cytokine assays in Cerrotini culture medium (Dulbecco modified Eagle medium supplemented with 8% Knockout serum replacement, (Gibco, Lifetechnologies, Paisley, UK), 36 mg/l asparagine, 116 mg/l arginine, 10 mg/l folic acid, 1 g/l 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid, 0.05 mmol/l β-mercapto-ethanol, 100 U/ml penicillin, 100 mg/ml streptomycin and 1 μg/ml fungizone)) in presence or absence of 5 μg/ml hamster anti-mouse CD3 and hamster anti-mouse CD28 (BD biosciences) coated wells. After 3 days of stimulation, culture supernatants were collected and frozen at − 80 °C prior to cytokines assay. Cytokines were measured in supernatant of primary cell culture of spleen or siLP by ELISA: IFN-γ and IL-17 present in primary cells culture supernatant were assayed using commercial enzyme linked immunosorbent assays (ELISA kits; Duoset R&D Systems, Lille, France). Cytokines were measured in feces suspended in RIPA buffer (0.5% deoxycholate, 0.1% SDS and 1% Igepal in TBS) containing complete anti protease cocktail (Roche). Fecal protein concentrations were measured using BCA uptima kit (Interchim). Lipocalin was assayed using commercial ELISA kits (R&D Systems). Measurements were performed in duplicate and detection threshold is defined for each molecule by the manufacturer’s instruction.