Mast cell degranulation assay

5 × 10⁴ hMCs or 1 × 10⁵ mMCs were pretreated with BoNT A or B (0.5-5 pM) for 24 hours. After being washed 2 times with PBS, MC degranulation was assessed by measuring the activity of β-hexosaminidase in the supernatants[39-41] of 1 × 10⁵ mMCs in 200 μl Tyrode’s buffer (0.1% BSA, 0.1% glucose, 2 mmol/l MgCl₂, 137.5 mmol/l NaCl, 12 mmol/l NaHCO₃, 2.6 mmol/l KCl, pH 7.4) or 5 × 10⁴ hMCs in 100 μl saline phosphate buffer (0.9 % NaCl, 10 mM NaH₂PO₄, 45 mM glucose) incubated for 30 min at 37°C with Compound 48/80 (10 μg/ml, Sigma) which was used to promote MC degranulation in an IgE-independent manner[42]. For each sample assayed, MC supernatant aliquots (20 μl) were mixed with substrate solution (100 μl), which consisted of 1 mM 4-methylumbelliferyl-2-acetamide-2-deoxy-β-D-glucopyranoside (Calbiochem) in 0.1 M sodium citrate buffer (pH 4.5), and were incubated for 2 hours at 37°C in the dark. The reaction was then stopped by the addition of 12 μl of 0.2 M glycine (pH 10.7). The reaction mixtures were excited at 365 nm and measured at 460 nm in a fluorescence plate reader (Gemini EM microplate spectrofluorometer, Molecular Devices). To determine the total cellular content of this enzyme, an equivalent number of cells were lysed with 1% triton-X-100 (Sigma). Release of β-hexosaminidase was calculated as the percentage of the total enzyme content.