Human il 13 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.

The blood samples were centrifuged for 10 min at 1500× g to obtain plasma samples which were kept at −80 °C for subsequent cytokine analysis. The levels of IL-8, IL-18, IL-23, IL-4, TGF-β, IL-22, IL-1β, IL-10, IL-15, CCL20, CCL2, CCL5, CX3CL1, and BDNF were measured by DuoSet^® ELISA Kits (R&D Systems, Minneapolis, MN, USA). The IL-13 levels were measured by the Human IL-13 ELISA kit (Invitrogen, Thermo Fisher, Waltham, MA, USA). High-sensitivity kits were required to evaluate IL-6 (Human IL-6 Quantikine HS ELISA Kit, R&D Systems, Minneapolis, MN, USA). The concentrations of TNF-α, IL-12p70, IL-17A, and IFN-γ were measured with cytokine 6-plex Panel 1 (TNF-α, IL-12p70, IL-17A, IL-10, IL-6, and IFN-γ) (IFN-γ, IL-6, IL-10, IL-12p70, IL-17A, TNF-a) (Quanterix Corp., Billerica, MA, USA) using SIMOA SR-X equipment (Quanterix Corp., Billerica, MA, USA). Additional measurements for IL-17A were performed with the IL-17A 2.0 Advantage Assay using SIMOA™ HD-X equipment (Quanterix Corp., Billerica, MA, USA).

CD4⁺ T lymphocytes were isolated from frozen PBMC using the EasySep™ Human CD4⁺ T Cell Isolation Kit (Stem Cell Technologies, Vancouver, BC, Canada). CD4⁺ cells were resuspended at a concentration of 1 × 10⁶ cells/mL in X-VIVO 20 serum-free medium (Lonza Group Ltd., Basel, Switzerland) supplemented with 1% penicillin/streptomycin. CD4⁺ cells (500,000 cells/well) were incubated in 48-well plates previously coated with an anti-CD3 antibody (Becton Dickinson, Franklin Lakes, NJ, USA) with or without a CD28 antibody (Becton Dickinson, Franklin Lakes, NJ, USA), which induces cell activation. After 6 h of incubation at 37 °C with 5% CO₂, the supernatant and cell pellets were collected for further analysis. The cytokines IL-17, IL-22, TNF-α, TFG-β, and IL-1β were measured in the culture supernatant by DuoSet^® ELISA Kits (R&D Systems, Minneapolis, MN, USA). For the IL-13 measurement, a human IL-13 ELISA kit was used (Invitrogen, Thermo Fisher, USA). IL-21 interleukin was measured in the cell pellet by western blot.