Anti dnp ige spe 7

BMMCs were incubated overnight at 37 °C with 100 ng/mL anti-DNP-IgE (SPE-7, Sigma-Aldrich) and then stimulated for 30 min at 37 °C with 100 ng/mL DNP-HSA antigen in 100 μL Tyrode's buffer. Control cells were treated with PBS. Culture supernatants were collected and cell pellets were lysed with 1% Triton X-100. To measure β-hexosaminidase activity, culture supernatant and cell lysates (30 μL) were incubated with 30 μL of substrate (1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide) for 60 min and then 0.1 M carbonate (250 μL) was added to stop the reaction. Absorbance of each compartment was measured at 405 nm. Percentage of β-hexosaminidase release was calculated as a percentage of total content, using the following formula: β-hexosaminidase release (%) = OD (supernatant)/[OD (supernatant)+OD (lysate)] × 100. For the hCBMCs degranulation assay, cells were incubated overnight with 100 ng/mL human myeloma IgE (Millipore, Temecula, CA, USA) and stimulated for 30 min with 100 ng/mL mouse-anti-human IgE (Invitrogen). Cells were then stained to confirm the surface expression of FITC-anti-LAMP-1 (H4A3) and PE-anti-hCD117 (104D2, Biolegend) by flow cytometric analysis.