Isolation and Culture of Mouse Eosinophils

The protocols, isolation, and culture of mouse eosinophils were fully described elsewhere^{25 (link)–28 (link)}. Generally, bone-marrow derived non-adherent mononuclear cells (NAMNCs) were seeded at 1 × 10⁶/ml in IMDM completed medium containing IMDM (Iscove’s modified Dulbecco’s medium; Invitrogen, Waltham, MA, USA) with 20% FBS (Gibco; origin from Australia), 100 IU/ml penicillin and 10 mg/ml streptomycin, 2mM L-glutamine, 1 × non-essential amino acids (Sigma-Aldrich, St. Louis, MO, USA), 1 mM sodium pyruvate (Sigma-Aldrich) and 0.006‰ β-mercaptoethanol (Sigma-Aldrich). 100 ng/ml rmFlt-3L (Peprotech, Rocky Hill, NJ, USA), and 100 ng/ml rmSCF (Peprotech) were supplemented from day 0 to day 4. Medium was replaced on day 4 and day 8, containing 10 ng/ml rmIL-5 (R&D Systems, Minneapolis, MN, USA), but without rmFlt-3L and rmSCF. Most experiments were performed in day 8 to day 10. Torin-1 (Tocris Biosciences) and U0126 (Selleck) were treated from day 4 when the medium was replaced. Cells were harvested and detected using PE-conjugated anti-SiglecF, and apoptotic levels were analyzed using AnnexinV-FITC and 7-AAD. Cells were lysed and detected by western blotting with p-S6 (Cell signaling technology, Denver, MA, USA), LC3B, Erk1/2, p-Erk1/2 and β-actin and analyzed Mbp and Gata-1 mRNA levels using quantitative real-time PCR.