Generation of mouse bone marrow-derived eosinophil (BmEOS) and DQ-OVA uptake by BmEOS
The ex vivo culture of BmEOS was adapted from the previously published protocols with minor modifications21, 22. Briefly, Bone marrow (BM) was flushed out from the tibia and fibula 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. Cells were evaluated by flow cytometry and performed from day 10 to 14.
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 (Themofisher, USA) for 6 hours before being collected, washed and further analyzed using flow cytometry. As a negative control, cells were cultured in the presence of DQ-OVA on ice.