Anti dinitrophenyl dnp ige clone spe 7

Mast cell degranulation was evaluated by the β-hexosaminidase release assay [28 (link)]. In brief, bone marrow derived mast cells (BMMCs) were sensitized at 1 × 10⁶/mL in the presence or absence of sunitinib in complete RPMI 1640 supplemented with 1.5 µg/ml anti-dinitrophenyl (DNP) IgE (clone SPE-7, Sigma-Aldrich) for 2 hours at 37°C in 5% CO₂. Cells were then washed once in Tyrode’s buffer (130 mmol/L NaCl, 10 mmol/L HEPES, 1 mmol/L MgCl₂, 5 mmol/L KCl, 1.4 mmol/L CaCl₂, 5.6 mmol/L glucose, and 0.05% bovine serum albumin, pH 7.4) and resuspended at 2 × 10⁶/mL in Tyrode’s buffer. Cells were then stimulated with recombinant murine SCF (10 ng/mL, PeproTech) for 5 minutes at 37°C. After the cells were spun down, 30 µL of supernatant was transferred to a 96-well flat-bottom plate. Then 30 µL of 1 mmol/L p-nitrophenyl-N-acetyl–D-glucosamide was added to each supernatant and mixed before incubation for 1 hour at 37°C. The reaction was terminated by the addition of 200 µL of 0.1 M Na₂CO₃-NaHCO₃ buffer, and optical density was read on a plate reader at a wavelength of 405 nm.