Human ige

Human PBCMCs (10⁵ cells/sample) were pre-sensitized with 1 μg/ml human IgE (Millipore) overnight, washed and stimulated with rabbit anti-human IgE (Bethyl), SCF variants or a combination of anti-IgE and SCF variants in 100 μl Tyrode’s buffer for 30 minutes. The concentrations of anti-IgE tested were 2000, 200, 20, or 2 ng/ml while those of SCF variants were 1000, 100, 10, or 1 ng/ml. For the combination stimulation, IgE pre-sensitized PBCMCs were treated with 1000, 100, 10 or 1 ng/ml of SCF variants in the presence of 2 ng/ml anti-IgE. The cells were then solubilized with 0.5% Triton X-100 in Tyrode’s buffer. The amount of β-hexosaminidase in supernatants and in the insoluble cell pellets were measured with p-nitrophenyl N-acetyl-β-D-glucosaminide in 0.1 M sodium citrate (pH 4.5). The reaction was stopped by adding 0.2 M glycine (pH 10.7). The release of the product 4-p-nitrophenol was detected at 405 nm. The extent of degranulation (i.e. β-hexosaminidase release) was calculated as the percentage of 4-p-nitrophenol absorbance in the supernatants, over the sum of absorbance in the supernatants and in cell pellets (total β-hexosaminidase) solubilized in detergent.

For TNF release, LAD2 cells (1×10⁵) were pre-incubated with cromolyn, lut or methlut (10–100 μM) and subsequently stimulated with SP (2 μM) for 30 min to measure preformed TNF release. LAD2 cells were also stimulated for 24 h to measure de novo synthesized TNF release, which occurs in addition to the rapid preformed TNF release . TNF was measured in the supernatant fluids using a TNF ELISA assay kit (R&D Systems, Minneapolis, MN).
For CCL2 release, primary hCBMCs (1×10⁵) were primed with human IgE (1 μg/mL, Millipore) overnight and pre-incubated with lut or methlut (50 μM) for 30 min before stimulation with anti-IgE (10 μg/mL, 2 hr, Life technologies). CCL2 was measured using a CCL2 ELISA assay kit (R&D Systems).