Legendplex bead based assay

Bulk LPLs or sort-purified ILC2s were incubated in DMEM with high glucose supplemented with 10% FCS, 10 mM Hepes, 1 mM sodium pyruvate, non-essential amino acids, 80 μM 2-Mercaptoethanol, 2 mM Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin (all from Gibco) in 96-well microtiter plates (Corning) for 4h at 37°C and 5% CO₂. Neuromedin U-23 (Phoenix Pharmaceuticals or Alpha Diagnostics) or a control peptide (Alpha Diagnostics) were added at 0.1 (Fig, 2 e, f, h), 1 μg/ml (Fig, 2 a-c, g, i,) or 10 μg/ml (Fig, 2 j-o) if not otherwise indicated. If indicated, the culture was supplemented with phorbol 12-myristate 13-acetate (PMA, 1 μg/ml) and ionomycin (Sigma-Aldrich, 1 μg/ml), IL-2, IL-7, IL-33 (R&D, 100 ng/ml, each) and/or IL-25 (eBioscience, 100 ng/ml). The inhibitor of Gαq proteins FR900359 was purified at the University of Bonn and used at 1 μM concentration. In experiments in which intracellular cytokine staining was performed, brefeldin A was added (Sigma-Aldrich, 10 μg/ml).
Cytokines in the supernatant were detected with a sandwich ELISA using IL-5 (TRFK5) or IL-13 (eBio13a) as capture antibodies and IL-5 (TRFK4) or IL-13 (eBio1316H) as detection antibodies (all from eBioscience) or the Legendplex bead-based assay (Biolegend) for IL-5, IL-9 and IL-13 according to the manufacture’s protocol.