Isolation and Culture of Immune Cells

Blood was collected by cheek bleeding or intracardial puncture. Spleens were smashed through a 70 μm nylon mesh filter (Becton Dickinson). Primary BM cells were harvested by flushing femurs' content using IMDM (Gibco). Red blood cells were lysed in an ammonium chloride buffer, and peritoneal lavages were harvested, as described elsewhere (29 (link)). For skin cell preparations, ears were split into the dorsal and ventral layers, minced and digested 30 min at 37C in a shaking incubator (150 rpm) in IMDM 5% FCS (Gibco) containing 2 mg/mL of collagenase IV and 100 μg/mL DNAse I (both Sigma). Digestion was stopped by adding 5 mM EDTA and a single cell suspension obtained by smashing the remaining tissue through a 70 μm nylon mesh filter (BD). Cells were cultured ex vivo in DMEM 20% FCS + Non-essential amino acid + Sodium pyruvate (Gibco) at 37C +5% CO₂. Peritoneal lavages incubation with DT was done in the presence of 10 ng/mL of Stem cell factor (SCF) (Peprotech). Whole splenocytes were stimulated with IL3 (Peprotech) or sterile-filtered FcBlock (2.4G2, Becton Dickinson, 10 μg/mL), MAR1 (0.5 μg/mL) or Ba13-APC (Biolegend, 1 μg/mL), Phorbol–myristate acetate (PMA, 100 ng/mL), ionomycin (100 ng/mL) or both (Thermofischer) for the indicated times in culture medium.