Anti fcεriα clone mar 1

Ears were harvested and separated into dorsal and ventral halves using forceps. For epidermal removal, the skin was incubated epidermis-down in 1.2 mg/ml Dispase II (Roche) at 37°C. After an hour, the epidermal layer was peeled off the dermis. To release cells, skin were torn into small fragments and digested for 1 h in 1 mg/ml collagenase D, 0.5 mg/ml type 2 hyaluronidase and 20 ug/ml Dnase 1 (all from Roche) at 37°C. Tissues were passed through a cell strainer and washed in PBS containing 3% FBS. Isolated cells were then stained for flow cytometry or cell sorting using anti-CD3 (clone 2C11, 1.0 µg/ml), anti-CD45R/B220 (clone RA3-6B2, 1.0 µg/ml), and anti-CD117 (cKit clone 2B8, 1.25 µg/ml) antibodies from BD Pharmingen, and anti-CD45.2 (clone 104, 0.5 µg/ml), anti-FcεRIα (clone MAR-1, 0.5 µg/ml) antibodies and streptavidin PE (0.4 µg/ml) from eBioscience, and anti-CD11c (clone N418, 2.5 µg/ml) from BioLegend. MCs were gated as CD45.2⁺, CD3⁻, B220⁻, CD11c⁻, cKit⁺ and FcεR1α⁺. For mRNA isolation, sorted MCs were directly collected into lysis buffer (Bioline ISOLATE II RNA Micro Kit).