RBL-2H3 cells (5 × 104) were allowed to settle onto activating or nonactivating surfaces in a total volume of 100 μl of Hepes buffer (10 mM Hepes, 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4·7H2O, 5.6 mM glucose, 1.3 mM MgSO4·7H2O, and 0.04% BSA) for 30 min at 37°C without CO2. A sample of supernatant (75 μl) was removed and centrifuged at 11,000g for 1 min in a 1.5-ml tube to remove any cells. Of this, 50 μl was then transferred to a well of a transparent, flat-bottomed 96-well plate. Negative control wells containing only Hepes buffer were also plated. We then added 100 μl of p-nitrophenyl N-acetyl-β-d-glucosamide (3.5 mg/ml in 40 mM citric acid) and 20 mM Na2HPO4·7H2O (pH 4.5) to each well and incubated the samples for 90 min at 37°C without CO2. The reaction was stopped after 90 min by the addition of 50 μl of 400 mM glycine (pH 10.7). Absorbance at 405 nm was measured using a SPECTROstar Nano plate reader. Background was established relative to the wells containing Hepes buffer alone. The percentage degranulation was calculated by reference to the absorbance at 405 nm of RBL-2H3 cells solubilized with 0.1% Triton X-100, which was defined as 100%.

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