Mast Cell Histamine Release Assay

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.