Recombinant il 25

The serum samples were achieved through 15 minutes centrifugation of 10 ml venous blood at 1000 × g. Ficoll density gradient was used for the purification of peripheral blood mononuclear cells (PBMC) from the blood of AR and controls.
Purified PBMCs (10⁶/mL) from AR patients were activated by phorbol myristate acetate (PMA, Sigma-Aldrich), ionomycin (Sigma-Aldrich), and Brefeldin A (Sigma-Aldrich) for 3 hours. Then, the ILC2s were sorted through staining by FITC lineage-negative cocktail kit, CD45, CRTH2, and CD127 (BD Bioscience). In detail, the lineage negative (Lin⁻) cells were obtained using the FITC lineage cocktail (CD2, CD3, CD14, CD16, CD19, CD56, and CD235a; BD Bioscience) and FceRI (eBioscience). Then, the Lin⁻ cells were stained with CD45, CRTH2, and CD127 (BD Bioscience) for isolation of ILC2s. Finally, the ILC2 was sorted using a FACS ARIA flow cytometer (BD Biosciences) with a purity of greater than 93%. Then, the data were analyzed by FlowJo software (TreeStar).
Sorted ILC2s (Lin⁻ CD45⁺CRTH2⁺CD127⁺) were incubated in 96-well plates with a density of 2.5 × 10⁵ cells/mL. The culture medium contained recombinant IL-25, IL-33, TSLP (all 10 ng/mL), IL-2 (50 ng/mL), or rhIL-37b (1–100 ng/mL) (all from the R&D Systems) for 72 hours. The ILC2s' proliferation was detected using the incorporation of tritiated thymidine.