Plate bound anti cd3 antibody

Unless otherwise stated, cells were cultured in RPMI 1640 medium (Gibco, New York, USA) containing 10% foetal bovine serum (FBS; HyClone, Los Angeles, USA), 1% 1 M HEPES, 2 mm glutamine (Sigma‐Aldrich, Milwaukee, USA), 100 U mL⁻¹ penicillin (Sigma‐Aldrich), 0.1 mg mL⁻¹ streptomycin (Sigma‐Aldrich), 1% 100 × sodium pyruvate and 2 μm 2‐mercaptoethanol (Invitrogen, Carlsbad, USA) in a humidified atmosphere (37°C and 5% CO₂). Activated CD4⁺ T cells were purified magnetically from splenocytes isolated from wild‐type C57BL/6 mice aged 6–8 weeks (purity > 90%) using a CD4⁺ T‐cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured at 1 × 10⁶ per well in the presence of plate‐bound anti‐CD3 antibody (2 µg mL⁻¹, Biolegend, California, USA) and soluble anti‐CD28 antibody (2 µg mL⁻¹, Biolegend) for activation.⁵⁶ Inactive CD4⁺ T cells were cultured in the absence of anti‐CD3 and anti‐CD28 antibodies. Different concentrations of Pg or LGG bacterial extracts were added to the culture medium to stimulate cells for subsequent detection via flow cytometry, RT‐qPCR or enzyme‐linked immunosorbent assay (ELISA).