Abstract
RBL-2H3 rat mast cells were stimulated to secrete intracellular granules through multivalent binding of antigen to receptor-bound IgE and preferentially used for anti-allergic evaluation. β-Hexosaminidase stored in the granules was used as a marker of mast cell degranulation. We developed a simple screening method to evaluate inhibitory effects of mast cell degranulation by efficient measurements of the enzyme activity in a continuous 96-well format. The cells were inoculated into a 96-well plate (2.5×105/well) and cultured overnight. Anti-dinitrophenyl-IgE, food extracts and dinitrophenyl-human serum albumin were successively added and incubated for each optimized interval of time. The supernatant was transferred to another well, and the cells were lysed with a passive lysis buffer instead of conventional sonication. β-Hexosaminidase activity in the supernatant and the cells was measured using a color formation substrate with a microplate reader. The percentage of degranulation was calculated from the ratio of the enzyme activity (supernatant/cells). The proposed method can be easily completed by a multi-channel pipetting operation and adapted for high-throughput screening (21 samples/plate, n=2) as compared to the conventional 24-well based method (3 samples/plate, n=2). Reproducibility was also improved from RSD 12.3% to 5.4-9.0% (within-run, n=4). The results agreed well with those of the previous method.
