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.