Degranulation Assay

LAD2 cells, primary human skin-derived mast cells and mouse peritoneal cells were resuspended in 0.1% SIR-BSA containing different concentrations of osthole. After 30 min, 45 μL of cells (0.45 × 10⁶ cells/mL for LAD2 and skin mast cells and 2 × 10⁶ cells/mL for mouse peritoneal cells) were seeded per well of 96-well plate and stimulated with compound 48/80, substance P, LL-37 or (R)-ZINC-3573 for 30 min. For assays using RBL-2H3 cells expressing MRGPRX2, 45 μL (1.2 × 10⁶ cells/mL) of cells were plated per well of a 96-well plate and treated with different concentrations of osthole and the MRGPRX2 agonists as described above. Supernatant were collected and incubated with an equivalent volume of 4 mM p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG, Sigma-Aldrich) for 1 h at 37°C. The reactions were halted through the addition of 0.1 M NaHCO₃/0.1M Na₂CO₃ buffer. Absorbance was measured using FlexStation^® 3 multi-mode plate reader (Molecular Devices; San Jose, CA, United States) at 405 nm. The total β-hexosaminidase content was measured by lysing cells with 0.1% Triton X-100 and then incubating the supernatant of the lysed cells with PNAG. Percent β-hexosaminidase release content was calculated by dividing the absorbance of agonist-stimulated cells by total cell β-hexosaminidase content. The spontaneous β-hexosaminidase release for LAD2, RBL-2H3 and primary human skin-derived mast cells was between 5 and 20% and for mouse peritoneal cells was between 4 and 20%.