Mouse il 5 elisa kit

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.

Antigen re-stimulated and control splenocytes were incubated in the presence of 2 µg/mL anti-mouse CD28 antibody for 60 h at 37 °C and 5% CO₂. The culture media were harvested and the IL-5 cytokine secretion was analyzed using a Mouse IL-5 ELISA kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The background values of unstimulated cultures were subtracted for each individual mouse.