Spe 7

WT mice were sensitized by subcutaneous injection into the ear lobe with 1.25 μg of anti-DNP-specific IgE (SPE-7; Sigma-Aldrich). Mice were then challenged 24 h later with 20 μl of 0.2% 2,4-dinitrofluorobenzene (DNFB; Nacalai Tesque, Kyoto, Japan) in acetone:olive oil (4:1) or vehicle alone. Ear tissues were collected 10 days after challenge for histological examination.

BMMCs were sensitized with 1 µg/mL of anti-DNP IgE (SPE-7, Sigma) in culture medium for 18 h, and then treated with 20 ng/mL of DNP-BSA for 20 min to activate them. Degranulation of BMMCs was assessed by flow cytometric measurement of the surface expression of CD107a (Lamp1). In some experiments, degranulation was evaluated by measuring the release of β-hexosaminidase upon stimulation as well. After washing with Tyrode’s buffer solution (10 mM HEPES, 130 mM NaCl, 6.2 mM D-Glucose, 3.0 mM KCl, 1.4 mM CaCl₂, 1.0 mM MgCl₂ and 0.1% BSA), sensitized BMMCs (1 × 10⁵ cells in 100 µL of Tyrode’s buffer solution/well in 96-well plate) were stimulated with DNP-BSA for 30 min, and then 50 µL of supernatants were incubated for 60 min with equal volume of 1 mM p-nitrophenyl-N-acetyl-p-d-glucosamine (Sigma) in 0.1 M sodium citrate buffer (pH 4.5) at 37 °C. The reaction was terminated by the addition of 0.2 M glycine (pH 10.7) and the released product, 4-p-nitrophenol, was detected by absorbance at 405 nm. The degranulation value was calculated by dividing 4-p-nitrophenol absorbance in the supernatant by the absorbance in detergent-solubilized unstimulated cell pellet.