Stem cell factor (scf)

Mice were housed in the animal facilities at the Istituto Superiore di Sanità (Rome, Italy). All experiments and procedures were approved by the Italian Ministry of Health (Permit No. 243/2016-PR to protocol No. D9997.13) and the Ethical Committee for Animal Experimentation of the Istituto Superiore di Sanità, in accordance with the Guide for the Care and Use of Laboratory Animals of the European Community Council Directives. Bone marrow-derived murine eosinophils were obtained following a protocol already described (Andreone et al., 2019 (link)), with some modifications. Briefly, bone marrow cells from naïve C57Bl/6 mice were cultured in RPMI 1640 containing 20% FBS, 1% glutamine, 25 mM Hepes, 1X NEAA, 1 mM sodium pyruvate, supplemented with 100 ng/mL SCF and 100 ng/mL FLT3-L (all from Cell Guidance Systems). From day 4, 10 ng/mL IL-5 (Peprotech) was added to the culture every other day. At day 14, eosinophils were harvested and re-plated at the concentration of 1,5 × 106/mL in the presence of either 100 ng/mL IL-33 (Biolegend; activated EOS) or 10 ng/mL IL-5 (resting EOS). GM-CSF (10 ng/mL; Cell Guidance Systems) was added to the cultures 24 h prior to use (day 15 or 16). Eosinophil purity (>80%) was determined by flow cytometry (CD11b⁺Siglec-F⁺Ly6G⁻CD11c⁻).