Rabbit anti 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.