Mouse il 33

Fresh lung ILC2s (CD45⁺Lin⁻CD127⁺CD90.2⁺ST2⁺) sorted from IL33-treated mice were cultured in 96-well round-bottom plates at a density of 5 × 10³ cells per well in RPMI-1640 medium containing 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin in the presence of 10 ng/ml human IL-2 (PeproTech), 20 ng/ml mouse IL-7 (PeproTech, 217-17-100), 1 ng/ml mouse IL-33 (BioLegend, 580506), 20 ng/ml human IL-7 (R&D Systems, 207-IL-010) and 50 ng/ml human IL-33 (R&D Systems, 3625-IL-010) with or without α-MSH (10 ng/ml) or β-endorphin (MCE, HY-P1502, 10 ng/ml). Then, the levels of cytokines in the culture supernatants were measured by ELISA (mouse IL-5 ELISA kit, Invitrogen, 88-7054-76; mouse IL-13 ELISA kit, Invitrogen, 88-7137-76; human IL-5 ELISA kit, Invitrogen, 88-7056-88; and human IL-13 ELISA kit, Invitrogen, 88-7439-88). For intracellular cytokine staining, 50 ng/ml PMA, 1 µg/ml ionomycin, and 1 µg/ml BFA were added to the cultures 2–4 h before staining.

Mouse IL-2 (catalog 575402), mouse IL-4 (catalog 547302), mouse IL-33 (catalog 580502), anti-mouse CD3ε (145-2C11), anti-mouse CD28 (37.51), human TGF-β1 (catalog 781802), anti-mouse IL-4 mAb (clone 11B11), anti-mouse IFN-γ mAb (XMG1.2), and Apotracker Green were purchased from BioLegend. Antibodies for TRAF4 (D1N3A), MyD88 (D80F5), p-mTOR (S2448, D9E), mTOR (7C10), p-AKT (S473, D9E), p-AKT (T308, D25E6), AKT (11E7), p-JNK (T183/Y185, 81E11), SAPK/JNK (no. 9252), p-p38 (T180/Y182, D3F9), p38 (D13E1), p-ERK1/2 (T202/Y204, D13.14.4E), Erk1/2 (137F5), p-IκBα (S32, 14D4), IκBα (L35A5), TRAF6 (D21G3), and β-Actin (8H10D10) were obtained from Cell Signaling Technology. Mouse T1/ST2 antibody (DJ8) and rabbit polyclonal against ST2 (ab228543) were obtained from MD Bioproducts and Abcam, respectively. AKT inhibitor VIII (CAS 612847-09-3) and LY3214996 (CAS 1951483-29-6) were purchased from Cayman Chemical. Primary mouse CD4⁺ T cells were cultured in complete TexMACS Medium (Miltenyi Biotec, 130-097-196) supplemented with 10% FBS, 50 μM β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin. CD8⁺ cells and ILCs were isolated from spleen using MojoSort Mouse CD8 T Cell Isolation Kit and Biotin anti-mouse Lineage Panel from BioLegend.