Mast cells stimulation and β-hexosaminidase release assay

LAD-2 cells and MC-9 cells were individually transferred to Tyrode’s solution (Jiandaoshou, Guangzhou, China) with A23187 calcium ionophore for 30 minutes for activation. At the end of the incubation, samples were centrifuged at 300 g for 5 minutes. Supernatants were collected to measure the β–hexosaminidase (β-Hex) release to assess LAD-2 cell activation. At the same time, equivalent cells were lysed with 0.5 %. Triton X-100, and total lysates were used to determine initial β-Hex content. Supernatants, total lysates, and controls were placed in 96-well plates (Corning, NY, USA). Substrate solutions (50 μl; p–nitrophenyl-N-acetyl-b-D-glucosaminide, 1.3 mg/ml in 0.1 mol/l citrate buffer, pH 4.5) were added to each well and incubated for 60 min at 37 °C (Jiandaoshou, Guangzhou, China). The reaction was stopped by adding 150 μl of 0.01 mol/l sodium carbonate/sodium bicarbonate (pH 10). β-Hex release was assessed by measuring sample optical densities (OD) at 405 nm using a microplate spectrophotometer and expressed as a percentage ratio.