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).