Dnp hab

Degranulation was estimated by measuring β-hexosaminidase release (Lian et al., 2015 (link)). Briefly, RBL-2H3 cells (2×10⁵ cells/well in 24-well plates) were sensitized with 0.2 μg/ml of monoclonal anti-dinitrophenyl specific mouse IgE (DNP-IgE, Sigma-Aldrich, D8406) overnight at 37°C in a 5% CO₂ incubator. To remove DNP-IgE before stimulation, cells were washed twice with PIPES buffer (pH 7.2), containing 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl₂, 0.1% BSA, and 1 mM CaCl₂. Cells were then incubated in 400 μl PIPES buffer with different concentrations of oroxylin A and incubated at 37°C for 30 min. Human dinitrophenyl albumin (1 μg/ml; DNP-hAb, Sigma-Aldrich, A6661) was then added, and cells were incubated for another 15 min at 37°C to induce degranulation. Aliquots (25 μl) of medium were then transferred to a 96-well microplate and incubated for 110 min with 25 μl of 5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich, N9376) in 0.1 M citrate buffer (pH 4.5). The reaction was terminated by adding 200 μl of 0.05 M sodium carbonate buffer (pH 10.0; 0.05 MNa₂CO₃/0.05 M NaHCO₃). Absorbance (OD) at 405 nm was measured using a microplate reader.

Degranulation was detected by measuring β-hexosaminidase release (Lee et al., 2016 (link)). Briefly, RBL-2H3 cells (1×10⁵ cells/well in a 24-well plate) were sensitized with 0.2 g/mL monoclonal anti-dinitrophenyl mouse immunoglobulin E (DNP-IgE, D8406, Sigma-Aldrich) overnight at 37°C in a 5% CO₂ incubator. Then, the cells were washed twice with piperazine-N,N′-bis-(2-ethanesulfonic acid) (PIPES) buffer (pH 7.2) containing 25 mM PIPES, 110 mM sodium chloride (NaCl), 5 mM potassium (KCl), 5.6 mM glucose, 0.4 mM magnesium chloride (MgCl₂), 0.1% bovine serum albumin (BSA), and 1 mM calcium chloride (CaCl₂) to remove the DNP-IgE. After incubation with different concentrations of PD1 at 37°C for 30 min, the cells were treated with 1 μg/mL human DNP-albumin (DNP-hAb, A6661, Sigma-Aldrich), and then incubated for an additional 30 min at 37°C to induce degranulation. Then, 25 μL of the supernatant was transferred to a 96-well microplate and incubated for 2 h with 25 μL 5 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide (N9376, Sigma-Aldrich) in 0.1 M citrate buffer (pH 4.5). The reaction was terminated by adding 200 μL stop buffer (0.05 M sodium carbonate [Na₂CO₃/0.05 M sodium bicarbonate [NaHCO₃], pH 10), and the optical density at 405 nm was measured.