Eosinophils isolation and culture
Peripheral blood eosinophils from healthy controls were purified by using an eosinophil isolation kit (Miltenyi Biotec, San Diego, Calif. USA, 130-092-010). Eosinophil purity was assayed using flow cytometry and Wright-Giemsa staining (Supplementary Fig. 2A). This procedure consistently resulted in a highly purified eosinophil population (95-99%). These eosinophils (>99% viable by trypan blue exclusion) were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml penicillin, 0.1 mg/ml streptomycin and 50 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF, Novoprotein, Suzhou, China, C003) at 37℃ in a humidified atmosphere of 5% CO2. Then, eosinophils (2x105/well in 200uL RPMI) were stimulated in a 96-well plate for 24 or 48 hours with or without the addition of the following agents: 50ng/mL recombinant TNF-α (Novoprotein, Suzhou, China, C008), 50ng/mL recombinant IL-5 (Novoprotein, Suzhou, China, CI59), 3 µM specific p38 MAPK inhibitor SB203580 (MedChemExpress, NJ., USA, HY-10256A). At the end of this incubation, eosinophils were harvested and investigated further by using flow cytometry for the expression of CD40 and ICOSL.