4.3. Degranulation Assay

LAD2 were treated with 50 µM resveratrol, quercetin or an equal volume of vehicle control (0.5% DMSO) for 24 h followed by sensitization with 100 ng/mL of human IgE (clone HE1, Invitrogen, Waltham, MA, USA) for 6 h or more. Cells were washed and resuspended in 10 mM HEPES buffer and transferred to a 96 well plate at a concentration of 2.5 × 10⁴ cells/well. Degranulation was induced with 10 µg/mL of anti-Human IgE (Invitrogen) for 30 min before measuring β-hexosaminidase (β-hex) release. The β-hex released into the supernatants and in cell lysates was quantified by hydrolysis of p-nitrophenyl N-acetyl-β-d-glucosamide (Sigma-Aldrich) in 0.1 M sodium citrate buffer (pH 4.5) for 90 min at 37 °C. The percentage of β-hex release was calculated as a percent of total content.

BMMC were seeded at 1 × 10⁶ cell/mL in a 24-well plate and sensitized with 0.5 µg/mL IgE (SPE-7; Sigma-Aldrich) and simultaneously treated with 0.1, 1 or 10 µM quercetin or resveratrol for 24 hr. BMMC were then washed and resuspended in 10 mM HEPES buffer and stimulated with 10 ng/mL DNP-BSA (Invitrogen) in a 96-well plate for 90 min. The β-hexosaminidase released into the supernatants and in cell lysates was quantified by hydrolysis of p-nitrophenyl N-acetyl-β-D-glucosamide (Sigma-Aldrich) in 0.1 M sodium citrate buffer (pH 4.5) for 60 min at 37 °C. The percentage of β-hexosaminidase release was calculated as a percent of total content.