Biotinylated human ige

Anti-MITF (clone D5G7V) and phospho-p44/42 MAPK antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-MITF (Ser73/180) and mouse anti-αTubulin antibodies were from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-Lamin β1 antibody was purchased from Abcam (Cambridge, UK). PE anti-human FcϵRI antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-LysRS (D-4) and PE anti-human CD117 were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). PE anti-human MRGPRX2 was from Bio Legend (San Diego, CA, USA ). APC anti-human CD63 and FITC Annexin V were from ImmunoTools GmbH (Friesoythe, Germany). Biotinylated human IgE was from Abbiotec (San Diego, CA, USA), and streptavidin was from Sigma (St. Louis, MO, USA). Substance P was from AnaSpec (Fremont, CA). ML329 was obtained from Axon Med Chem (Groningen, The Netherlands). Morphine was obtained from B. Braun Medical S.A (Spain), the muscle relaxant atracurium was from Pfizer Inc (NY, USA), meglumine amidotrizoate was from Juste Laboratories (Spain), and the vancomycin antibiotic was from Normon (Spain).

For IgE-dependent activation, cells were sensitized overnight with 0.1 mg/mL biotinylated human IgE (Abbiotec, San Diego, CA, USA) and stimulated with 0.4 mg/mL streptavidin (Sigma, St. Louis, MO, USA) for 30 min at 37 °C. Cells were blocked and stained with PE-conjugated anti-CD63 (BD Biosciences, Mountain View, CA, USA) and propidium iodide to exclude dead cells. Cells were acquired on a FACS Calibur flow cytometer (FACScan; BD Biosciences, Mountain View, CA, USA) and analyzed using FlowJo 7.6 software.