U bottom culture plates

Cells derived from the MLN were cultured in U‐bottom culture plates (Greiner, Frickenhausen, Germany) at 8 × 10⁵ cells per well in culture medium (RPMI 1640 medium (Lonza, Verviers, Belgium) with 10% FCS, penicillin (100 U/mL)/streptomycin (100 µg/mL, Sigma)). Cells were stimulated with culture medium as a negative control, a polyclonal stimulation with anti‐CD3/CD28 (1 µg/mL, clone 145‐2C11 and clone 37.51, eBioscience) or antigen‐specific stimulation with PE (100 µg/mL). To assess production of Interleukin (IL)‐5, IL‐10, IL‐13, and Interferon‐γ (IFN‐γ) by T cells, cells were incubated for 48 h (anti‐CD3/CD28) or 96 h (PE). Culture supernatants were collected and stored at −20°C until further analysis with the Ready‐SET‐Go!® ELISA (eBioscience) according to the manufacturer's instructions.

8 × 10⁵ cells per well in 200 μl derived from spleen and MLN (in CMA model) were cultured in U-bottom culture plates (Greiner, Frickenhausen, Germany) using RPMI 1640 medium (Lonza, Verviers, Belgium) with 10% FCS, penicillin (100 U/ml)/streptomycin (100 μg/ml) (Sigma) and β-mercaptoethanol (CMA model, 20 μM). All cells received either stimulation with culture medium as a negative control, a polyclonal stimulation with anti-CD3 (CMA model; 1 μg/ml, clone 17A2, eBioscience) or anti-CD3/CD28 (PNA model; 1 μg/ml, clone 145-2C11 and clone 37.51, eBioscience) or antigen-specific stimulation with whey (50 μg/ml) or PE (100 μg/ml). Plates were incubated for 48 h (anti-CD3 or anti-CD3/CD28) or 96 h (whey or PE) to assess production of interleukin (IL)-5, IL-10, IL-13 and Interferon γ (IFNγ) by T cells. Culture supernatants were collected and stored at −20 °C until further analysis with the Ready-SET-Go!^® ELISA (eBioscience) according to the manufacturer’s instructions.