Bwa4c

Bone marrow-derived basophils (BMBAs) and bone marrow-derived mast cells (BMMCs) were generated as described previously [20] (link), [21] with minor modification. In brief, mouse bone marrow cells were cultured with 10 ng/mL murine IL-3 and 2 ng/mL murine SCF for 40 days to obtain BMMCs while they were cultured with 0.1 ng/mL IL-3 for 7 days to obtain BMBAs. In case of BMBAs, the basophil fraction in cultured bone marrow cells was purified with positive sorting of CD49b⁺ cells by using MACS pro system (Miltenyi Biotec). The purity of basophils (CD49b⁺CD200R3⁺cKit^-) in the BMBA preparation and mast cells (CD200R3⁺cKit⁺) in the BMMC preparation was >90%. Identity of basophils and mast cells was verified by their selective expression of Mcpt8 and Mcpt6 mRNAs encoding serine proteases mMCP-8 and mMCP6, respectively [20] (link), [22] , [23] . BMBAs and BMMCs were sensitized overnight with 1 μg/mL of hapten 2,4,6-trinitrophenyl (TNP)-specific IgE (IGELb4, ATCC-TIB141), and then incubated with 10 ng/mL TNP-conjugated ovalbumin (TNP₁₂-OVA, Biosearch Tech.) or control ovalbumin (OVA, Invivogen) for indicated time periods. In some experiments, before stimulation, cells were pretreated for 10 min with of 3 μmol/L of inhibitors, including BW-A4C (5-LOX inhibitor, Sigma), SC-560 (COX-1 inhibitor, Cayman Chemicals), and celecoxib (COX-2 inhibitor, Sigma).