Mast Cell Degranulation Assay with RBL-SX38 Cells

Human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells transfected with a nuclear factor of activated T-cells (NFAT)-responsive luciferase reporter gene, were used to measure mast cell degranulation as previously described [29 (link)]. In short, these RBL cells were plated in clear bottom 96 well plates and sensitized using an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG, a major allergen in bovine whey), as described by Knipping et al. [30 (link)]. Hereafter, RBL cells were incubated for 24 h with 0.05 and 0.5% scFOS/lcFOS (ratio 9:1). To induce degranulation, cells were exposed to 1, 10, 100 and 1000 ng/ml BLG. After stimulation, luciferase substrate solution containing cell lysis reagent (One-Glo, Promega Corp., Tokyo, Japan) was added to the cells, and chemiluminescence was measured. Luciferase expression levels are represented as the fold increase of relative light units compared with the background expression, after subtraction of a blank control (without cells).