Biotin anti il 4

Freshly isolated spleen-derived DO11.10 CD4⁺ T cells at 0.2
× 10⁶ cells per well on 24-well plates were cultured with 0.2
× 10⁵ BMDCs and 20 mg/ml chicken OVA for 72 h. In other cases,
20 μg/ml chicken OVA peptide (323–339) (LifeTein) was used. In
some cases, 30 ng/ml IL-12 or 10 ng/ml IL-6 (PeproTech), 10 ng/ml IL-23, and 10
ng/ml IL-1β (eBioscience) were also added. The following were assessed by
ELISA: anti– IFN-γ, biotin anti–IFN-γ,
anti–TGF-β, biotin anti–TGF-β, anti–IL-10,
anti–IL-2, and biotin anti–IL-2 (from BD Biosciences);
anti–IL-17, biotin anti–IL-17, anti–IL-5, biotin
anti–IL-5, anti–IL-13, and biotin anti– IL-13 (from
eBioscience); and anti–IL-4, biotin anti–IL-4, and biotin
anti–IL-10 (from BioLegend). Cell proliferation was assessed with MTT as
per manufacturer instructions (MilliporeSigma).

Freshly isolated spleen-derived DO11.10 CD4⁺ T cells from IRF5^+/+, IRF5^+/−or IRF5^−/−mice at 0.2×10⁶ cells/well on 24 well plates were stimulated with 5 μg/ml anti-CD3 (145–2C11, Bio X Cell, West Lebanon, NH) and 1 μg/ml anti-CD28 (PV-1, Bio X Cell) antibody-coated wells for 72 hours. The following were assessed by ELISA: from BD biosciences: anti-IL2, biotin anti-IL2, anti-IFNγ, biotin anti-IFNγ, anti-TGFβ, biotin anti-TGFβ; from ThermoFisher Scientific: anti-IL17, biotin anti-IL17, biotin anti-IL5, anti-IL13, biotin anti-IL13; from BioLegend: anti-IL4, biotin anti-IL4, anti-IL5, anti-IL10, biotin anti-IL10. MTT assay as per manufacture instructions (MilliporeSigma).