Bioline isolate 2 rna micro kit

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).

RNA was extracted using TRIzol (Life Technologies, VIC, Australia) and subsequent steps were performed as per the Bioline Isolate II RNA Micro kit manufacturer’s instructions (Bioline, Alexandria, NSW, Australia). cDNA was synthesized using the SensiFAST^TM cDNA Synthesis kit from Bioline. SYBR Green Master Mix was used to perform qRT-PCR in a ViiA7 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) and analysis was performed using QuantstudioTM Real-Time PCR software v1.1 (Applied Biosystems, Life Technologies, VIC, Australia). The primer sequences are listed in Table S1.