Kusunose et al. the current presence of dexamethasone. The profiles of granule constituents were drastically modified by dexamethasone. Topical software of dexamethasone down-modulated secretagogue-induced degranulation and the expression levels of several Mrgpr subtypes in cutaneous cells. These results suggest that mast cell-mediated IgE-independent cutaneous swelling could be suppressed by steroidal anti-inflammatory medicines through the down-regulation of G i1 and several Mrgpr subtypes in mast cells. at 4 C for 5 min to obtain the supernatants (extracellular fractions, E). The resultant pellets were resuspended in PIPES-buffer comprising 0.5% Triton X-100 and were centrifuged at 10,000 for 10 min to obtain the supernatants (cell-associated fractions, C). Degranulation was evaluated by measuring enzyme activity of a granule enzyme, -hexosaminidase, in each portion, using the specific substrate, at 4 C for 30 min. The resultant supernatants were subjected to granule protease assays. Chymotryptic activity was measured in 33.3 mM Tris-HCl, pH 8.3 containing 3.3 mM CaCl2 and 0.3 mM gene family were analyzed by quantitative reverse transcription (RT)-PCR with DNase-treated Relugolix total RNAs. Total RNAs were prepared using NucleoSpin RNA kit (TaKaRa Bio, Kusatsu, Japan). PCR was performed using StepOne Plus (Thermo Fisher Scientific, Waltham, MA, USA) with KOD SYBR qPCR Blend (TOYOBO, Osaka, Japan) or Fast SYBR Green Expert Blend (Thermo Fisher Scientific, Waltham, Relugolix MA, USA) the specific primer pairs (ahead, reverse); 0.05, n = 3). Unexpectedly, enzymatic activity FAD of -hexosaminidase, a lysosomal enzyme, which might play a critical part in bactericidal action [19] and is often utilized for monitoring degranulation levels, was significantly up-regulated in CTMC-like MCs acquired in the presence of dexamethasone (Number 3b). Open in a separate window Number 1 Bone marrow-derived cultured mast cells (BMMCs) were co-cultured with Swiss 3T3 fibroblasts in the presence (closed circles) or absence (open circles) of 1 1 M dexamethasone for 16 days as explained in Materials and Methods. (a) The numbers of the cultured mast cells were counted on day time-0, 4, 8, 12, and 16. Ideals are offered as the means SEMs (n = 4). The ideals ** 0.01 are regarded as significant. (b) The ratios of the Safranin-positive cells were determined. Ideals are offered as the means SEMs (n = 4). Open in a separate window Number 2 BMMCs were co-cultured with Swiss 3T3 fibroblasts in the presence (closed circles or columns) or absence (open circles or columns) of 1 1 M dexamethasone for 16 days as explained in Materials and Methods. (aCc) Enzymatic activities of three kinds of granule proteases (a); chymotryptic activity, (b); tryptic activity, and (c); carboxypeptidase A activity) were measured. Ideals are offered as the means SEMs (n = 3). Ideals with * 0.05 and ** 0.01 are regarded as significant. (d) Manifestation levels of granule protease genes ( 0.05 (vs. D0) and # 0.05 (vs. D16, (?)Dex) are regarded as significant. Open in a separate window Number 3 (a,b) The cellular histamine material and enzymatic activities of -hexosaminidase in the mast cells co-cultured for 16 days in the presence (closed circles) or absence (open circles) of 1 1 M dexamethasone were measured. (cCf) The co-cultured mast cells were sensitized with IgE (1 g/mL, clone IgE-3) for 3 h and then stimulated with the indicated concentrations of the antigen, or stimulated with compound 48/80 (CP, 10 g/mL), compound P (SP, 100 M), or thapsigargin (Thg, 300 nM) without sensitization. Degranulation upon IgE-mediated antigen activation (c) and treatment with compound Relugolix 48/80, compound P, or thapsigargin (d) was measured in the mast cells co-cultured for 16 days in the presence (closed circles or columns) or absence (open circles or columns) of 1 1 M dexamethasone. (e,f) BMMCs were co-cultured for 16 days and were treated with 1 M dexamethasone during the last 24 h (closed circles and columns). Degranulation was then measured as explained above. (gCj) BMMCs were treated without (open circles or columns) or with 1 M dexamethasone (closed circles or columns) for 24 h. The cells were then sensitized with 1 g/mL IgE (clone IgE-3) for 3 h and stimulated with the indicated concentrations of the antigen or stimulated with thapsigargin (Thg, 300 nM) or A23187 (A23187, 1 M). Degranulation (g,h) and IL-6 launch (we,j) were measured. The degree of degranulation was determined by measuring -hexosaminidase activity. Ideals are offered as the.