Recombinant mouse neuromedin u 23 peptide nmu23

For in vitro experiments, purified lung and small intestine lamina propria ILC2s were cultured in complete RPMI (supplemented with 10% foetal bovine serum (FBS), 1% hepes, sodium pyruvate, glutamine, streptomycin and penicillin) at 37ºC. ILC2s were stimulated overnight for 2, 4, 6 and 20 hours with recombinant mouse Neuromedin U 23 peptide (NmU23) (100ng/mL unless stated otherwise; Phoenix Pharmaceuticals), recombinant mouse IL-25 and IL-33 (10ng/mL, unless stated otherwise) (R&D Systems). Control and activated ILC2s were cultured in the presence of IL-2 and IL-7 (10ng/mL; PeproTech), unless stated otherwise. ILC2s were lysed using RLT buffer (Qiagen). For cytokine protein analysis in vitro, ILC2s were incubated with brefeldin A (eBioscience) for 2, 4, 6 or 20 hours prior to intracellular staining. For in vivo experiments, mice were injected intraperitoneal (i.p.) with NmU23 peptide (8μg/day) during Nippostrongylus brasiliensis infection or with a single dose of NmU23 (20μg) and analysed after 12 hours. Control mice were treated with PBS alone. For cytokine protein analysis ex vivo, ILC2s were incubated with PMA (50ng/mL), ionomycin (500ng/mL) (Sigma) and brefeldin A (eBioscience) for 4 hours prior to intracellular staining.