Quantifying mast cell degranulation

LAD2 cells were washed twice and resuspended in 0.1% SIR-BSA. Fourty-five microliters of cells (0.45 × 10⁶ cells/mL) were seeded and stimulated with CST-14, LL-37, substance P, compound 48–80, or (R)-Zinc-3573 for 25 min. For total β-hexosaminidase release, cells were lysed using 0.1% Triton X-100. The supernatant (20 μL) was collected and incubated with an equivalent volume of 4 mM p-nitrophenyl-N-acetyl-β-D-glucosamine (PNAG) for 1 h at 37°C. The reactions were halted through the addition of 0.1 M NaHCO₃/0.1 M Na₂CO₃ buffer. The β-hexosaminidase release assay for human skin mast cells was performed as described previously by McHale et al. (39 (link)). For assays using inhibitors, cells were incubated for 30 min with the appropriate drug prior to agonist stimulations. Inhibitor concentrations were determined using IC50 values reported by the drug manufacturer. Absorbance was measured using FlexStation® 3 multi-mode plate reader (Molecular Devices; San Jose, CA) at 405 nm. Percent of β-hexosaminidase release content was calculated by dividing absorbances of agonist-stimulated cells by total cell β-hexosaminidase content.