Mast cell activation assay & Quantitative real-time PCR
Mast cells were cultured (if applicable) with JQ-1 (HY-13030, MedChemExpress) or sodium butyrate (303410, Sigma-Aldrich) 24 h prior to activation. PBCMCs from two different donors were sensitized with human IgE (2 μg/ml, for 1 hour) and washed, followed by stimulation with 2 μg/ml of anti-IgE. Human IgE myeloma was purchased from Milipore-Sigma (401152) and rabbit anti-human IgE from Bethyl Laboratories (A80-109A). Degranulation was measured by the release of beta-hexosaminidase 57. Substrate 4-MUG (M2133, Sigma-Aldrich) was added to this enzyme containing supernatant and the product was measured after 1 h by means of fluorescence (Glomax Discover, Promega). After RNA isolation as stated above, cDNA was made using RevertAid H Minus First Strand cDNA Synthesis Kit (#K1632, Thermo Fisher Scientific) according to manufacturer. qPCR is performed using PowerUp™ SYBR™ Green Master Mix (#A25741, Thermo Fisher Scientific) and primers were found by OriGene (Table 1). Run on QuantStudio™ 3 Real-Time PCR System (A28567, Thermo Fisher Scientific).