Preparation of bone marrow–derived mast cells (BMMCs), fetal skin–derived mast cells (FSMCs), and fetal liver–derived mast cells (FLMCs)

Bone marrow–derived mast cells (BMMCs) were generated from the femoral bone marrow cells of male mice as previously described⁵⁴. Fetal skin–derived mast cells (FSMCs) were generated from fetal skin of wild-type mice as previously described³¹. Fetal liver–derived mast cells (FLMCs) were generated from fetal liver cells from CLEC-2^−/− or CLEC-2^+/+ mouse¹³ embryos on embryonic day (E) 15.5. Briefly, the fetal liver was gently crushed through a fine mesh, and the resultant cells were cultured for 4–6 weeks in RPMI 1640 medium (Sigma-Aldrich) supplemented with 1 mmol/L sodium pyruvate (Gibco), penicillin–streptomycin (Gibco), non-essential amino acids (Gibco), 10% heat-inactivated FCS (Gibco), 100 µmol/L 2-mercaptoethanol (Kanto Chemical, Japan), 10 ng/mL of murine interleukin-3 (PeproTech), and 10 ng/mL of murine SCF (PeproTech). Mast cells were identified by flow cytometric detection cell-surface c-kit (CD117) and FcεRIα.