Isolation of Bone Marrow Eosinophils

Male WT and Ox40^-/- mice were used to obtain bone marrow eosinophils. After treatment with erythrocyte lysis buffer to remove RBCs, bone marrow cells were used for culture and sorting of mature BM eosinophils. Unselected mouse bone marrow progenitors harvested from WT or Ox40^–/– mice were cultured with recombinant mouse (rm)FLT3-L (100 ng/ml, 250-31L, PeproTech, USA) and rmSCF (100 ng/ml, 250-03, PeproTech) for 4 days, followed by culture with rmIL-5 (10 ng/ml, 215-15, PeproTech) alone thereafter for 4-6 days to generate bone marrow-derived eosinophils (bmEos) as previously described (22 (link)). On Days 8-10, cultured eosinophils were purified with a FACS Aria II cell sorter (BD Biosciences, CA, USA) using gating on 7AAD-Siglec-F+SSC-A^hi cells as previously described (22 (link)). For natural BM eosinophils, mature BM eosinophils were sorted as previously described (23 (link)). Briefly, B cells (anti-B220), T cells (anti-CD3), DCs (anti-CD11c), neutrophils and macrophages (anti-Gr-1) were depleted by MACS. Then, the remaining cells were incubated with anti-7AAD, anti-FcϵRIα, anti-Siglec-F, and anti-CD11b antibodies. Live mature eosinophils, gated as 7AAD-FcϵRIα-Siglec-F+CD11bint cells, were sorted with a FACS Aria II cell sorter.