Rmil 5

The ex vivo culture of BmEOS was adapted from the previously published protocols with minor modifications (21 (link), 22 (link)). Briefly, bone marrow (BM) was flushed out from the femur and tibiofibula of C57bl/6J mice. After erythrocyte lysis, BM cells were seeded at 1×10^6/ml in IMDM media supplemented with 20% fetal bovine serum (Gibco), 1% penicillin/streptomycin, 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Gibco), and 50 mM 2-mercaptoethanol (Sigma-Aldrich) in the presence of 100 ng/mL FLT-3L and 100 ng/mL recombinant murine stem cell factor (SCF, Peprotech), cultured from Day 0 to Day 4. On Day 4 and 8, the media containing SCF and FLT3-L was replaced with media containing 10 ng/mL recombinant mouse interleukin-5 (rmIL-5, R&D Systems) only. Every other day, from this point forward, media was replaced with fresh media containing rmIL-5. After Day 10, cells were seeded and used for subsequent experiments.
BmEOS were seeded in 48 wells plates and stimulated for 2 days with or without HDM and IL-25 in complete medium. To assess DQ-OVA uptake by BmEOS, BmEOS were treated with DQ-OVA for 12 h before being collected, washed, and further analyzed using flow cytometry. Cells cultured in the absence of DQ-OVA were set as a negative control.

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.