Human lineage cocktail

Mast cells were isolated from 6 mm biopsies obtained from 6 mm skin punch biopsies acquired from the buttocks of volunteers with and without psoriasis under local anaesthesia in the Salford Royal Foundation Trust Hospital Dermatopharmacology Unit. Biopsies were stored in ice-cold PBS. Skin biopsies were cut into small pieces in digestion solution containing RPMI 1640 medium (Sigma-Aldrich, Gillingham, UK), 0.5% BSA (ThermoFisher Scientific), 100 U.mL⁻¹ penicillin and 100 µg.mL⁻¹ streptomycin, 0.5 Wunch units.mL⁻¹ of Liberase TM (Roche, Basel, Switzerland), and 100 ng.mL⁻¹ of SCF (Genscript, Piscataway, NJ, USA), then incubated overnight at 37 °C, 5% CO₂.
Digested skin was passed through a 40 μm mesh cell strainer and cell concentration was adjusted to 50 × 10⁶ cells.mL⁻¹. Cell suspension was stained for 15 min in PBS 0.5% BSA, 2 mM EDTA with CD45 (2D1), Human Lineage cocktail (CD3 (UCHT1), CD14 (HCD14), CD19 (HIB19), CD20 (2H7) and CD56 (HCD56)), CD117 (104D2), FcɛRIα (AER-37) antibodies (all from BioLegend, San Diego, CA, USA), and a viability dye (Fixable Blue Dead Cell Stain Kit, ThermoFisher Scientific). CD45+, Lin-, CD117+, FcɛRIα+, and MCs were sorted using a BD Influx (BD Biosciences) (Supplementary Figure S3), collected in 1000 cell aliquots and stored at −80 °C until required.