Compound 48 80 c48 80

The HRA was performed according to a method routinely employed in our laboratory [34 (link),35 (link)]. In brief, MCs pretreated with or without inhibitors were stimulated by FcεRI-aggregation. The anti-AER-37 antibody (eBioscience, San Diego, CA, USA) was used for cultured MCs at 0.1 µg/mL, and the anti-FcεRIα-Ab 29C6 (kind gift from Dr. Hakimi, Hoffmann La Roche, Nutley, NJ, USA) at 0.5 µg/mL served for the stimulation of ex vivo MCs. MRGPRX2 stimulation was achieved by compound 48/80 (c48/80, Sigma, at 10 µg/mL), or substance p (SP, Bachem, Budendorf, Switzerland at 30 µM). Spontaneous release was determined in the absence of any stimulus. Assays were performed in PAG-CM buffer (Piperazine-N,N-bis [2-ethanesulfonic acid]-Albumin-Glucose buffer containing 3 mM CaCl₂ and 1.5 mM MgCl₂, pH 7.4) for 30 min at 37 °C. Histamine in the supernatants was measured by an automated fluorescence method (Alliance Instruments, Salzburg, Austria). Total cellular histamine content was measured analogously. All determinations were performed in triplicate. Net histamine release (%) was calculated as [(stimulated release–spontaneous release)/complete histamine in the MC preparation] × 100.

RPMI 1640 medium, Minimum Essential Medium (MEM), newborn calf serum, trypsin, collagenase I, and HEPES were purchased from Gibco (Thermo Fisher Scientific, Carlsbad, CA, USA). 4-Nitrophenyl N-acetyl-β-D-glucosaminide, compound 48/80 (c48/80), cromolyn sodium salt, penicillin, streptomycin sulfate, and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 was obtained from Dojindo Lab (Kumamoto, Japan). Histamine ELISA kit was purchased from IBL International (Hamburg, Germany). The CytoTox-ONE™ Homogeneous Membrane Integrity Assay kit was purchased from Promega (Madison, WI, USA). Percoll was purchased from Pharmacia (Stockholm, Sweden). Toluidine blue, glycine, and Triton X-100 were purchased from Sunshine Biotechnology Co. Ltd. (Nanjing, China). FITC-labeled rat mAb to c-Kit was purchased from Abcam (Cambridge, MA, USA). Goat anti-rat IgE polyclonal antibody was purchased from GeneTex Inc. (Irvine, CA, USA). Purified rat IgE was purchased from Life Technologies (Thermo Fisher Scientific, Carlsbad, CA, USA).

SHLI and its two intermediate fractions (F1, the extract of Scutellariae Radix; F2, the extract of Lonicerae Japonicae Flos and Fructus Forsythiae) were prepared by Duoduo Pharmaceutical Co., Ltd. (Jiamusi, Heilongjiang, China) according to the Chinese Pharmacopoeia¹ . Compound 48/80 (C48/80), propranolol, CV3988, triprolidine, globulins, cimetidine, and SB290157 were purchased from Sigma-Aldrich (St Louis, MO, USA). PMX53 was from GL Biochem Ltd. (Shanghai, China). Mouse tIgE ELISA kit was from Biolegend Co. (San Diego, CA, USA). Rehydragel^® aluminum adjuvant was from General Chemical (Parsippany, NJ, USA). Anti-human CD54-allophycocyanin (APC) antibody and its REA control-APC were from Miltenyi Biotec. (Bergisch Gladbach, German). Human C5a ELISA kit, fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD86 antibody and its isotype control were from BD Biosciences (San Diego, CA, USA). Gentamicin, ampicillin, 2-methyl-5-nitroimidazole-1-ethanol, and fradiomycin were from TCI Chemicals (Tokyo, Japan). Vancomycin was obtained from BBI Life Sciences Corporation (Shanghai, China). The mouse mast cell protease 1 (MMCP1) ELISA kit was from Invitrogen (San Diego, CA, USA). Protein G PLUS-Agarose was from Santa Cruz Biotechnology, Inc. (SantaCruz, CA, USA).

Lipopolysaccharide (LPS) and compound 48/80 (C48/80) were obtained from Sigma-Aldrich (St. Louis, MO). IL-6 and IL-10 ELISA kits were purchased from R&D Systems (Minneapolis, MN). The LPS and C48/80 stock solutions were prepared in pharmaceutical-grade sterile normal saline and subsequently diluted in sterile phosphate-buffered saline (PBS) as required.