Anti dnp ige clone spe 7

For mast cell activation, BMMCs were sensitized with 0.5 μg/mL anti–DNP-IgE (clone SPE-7; Sigma-Aldrich) overnight at 37°C at a density of 1 × 10⁶ cell/mL. Cells were washed, resuspended in Tyrode’s buffer, and challenged with 100 ng/mL of DNP-HSA (Sigma-Aldrich) and 2.5 μg/mL ionomycin (Sigma-Aldrich) for 30 minutes at 37°C. Mast cell activation was assessed by the analyses of β-hexosaminidase, histamine release, and CD107a (LAMP-1). Briefly, β-hexosaminidase was expressed as a percentage of β-hexosaminidase calculated from the total β-hexosaminidase in 0.5% Triton X-100 lysed BMMCs as previously described (53 (link)). Histamine release was measured after 30 minutes by taking the top 0.05 mL of culture supernatant, diluting in 1 mL acid solution for overnight protein precipitation, and assaying using automated fluorimetry as previously described (90 (link)). Expression of CD107a (LAMP-1) as one of the mast cell activation markers was detected by flow cytometry analysis. Intracellular ROS were detected using the oxidative sensitive fluorescent dyes CM-H₂DCFDA (Thermo Fisher Scientific) as previously reported (87 (link)).

UTP, ATP, ADP, PGE₁, PGE₂, 2,4-dinitrophenyl human serum albumin (DNP-HSA), anti-DNP IgE (clone SPE-7), p-nitrophenyl N-acetyl-β-d-glucosaminide, and the GenElute Mammalian Total RNA miniprep kit were obtained from Sigma-Aldrich (Tokyo, Japan). Allophycocyanin (APC)-conjugated rat anti-mouse c-Kit antibodies (clone 2B8) were obtained from BD Pharmingen (Tokyo, Japan). Phycoerythrin (PE)-conjugated mouse anti-mouse FcεRIα antibodies (clone MAR-1) were obtained from eBioscience (San Diego, CA, USA). Recombinant mouse interleukin (IL)-3 and recombinant mouse SCF were obtained from Peprotech (London, UK). MRS2179, AR-C118925, NF449, AZ10606120, 5-BDBD, Ivermectin, PP2, LY303511, LY294002, Triciribine, and AS605240 were obtained from Tocris Bioscience (Bristol, UK). R406 was obtained from Cayman Chemical (Michigan, USA). Fura-2-acetoxymethylester (AM) and PTX were obtained from Wako (Osaka, Japan). Anti-phospho-Syk, anti-Syk, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). ONO-DI-004 (selective EP1 agonist), ONO-AE1-259-01 (selective EP2 agonist), ONO-AE-248 (selective EP3 agonist), and ONO-AE1-329 (selective EP4 agonist) were obtained from ONO Pharmaceuticals (Osaka, Japan). All other chemicals were of reagent-grade or the highest quality available.