title: A microplate assay to assess chemical effects on RBL-2H3 mast cell degranulation: effects of triclosan without use of an organic solvent.
abstract: Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells(3-5). Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells(6), can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies(1). Originally published by Naal et al.(1), we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here. Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease(7-11), although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function(2). In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280 = 4,200 L/M/cm)(12). This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
authors.affiliation: Department of Molecular and Biomedical Sciences, University of Maine, Orono.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Weatherly
authors.forename: Rachel H
authors.initials: RH
authors.lastname: Kennedy
authors.forename: Juyoung
authors.initials: J
authors.lastname: Shim
authors.forename: Julie A
authors.initials: JA
authors.lastname: Gosse
mesh_terms: D000818:Animals; D000891:Anti-Infective Agents, Local / Q000494:pharmacology; D002021:Buffers; D015550:Cell Degranulation / Q000187:drug effects* / Q000276:immunology; D045744:Cell Line, Tumor; D003584:Cytological Techniques / Q000295:instrumentation / Q000379:methods; D015471:Leukemia, Basophilic, Acute / Q000473:pathology; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology / Q000502:physiology*; D051381:Rats; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D014260:Triclosan / Q000737:chemistry / Q000494:pharmacology*; D014867:Water / Q000737:chemistry
chemical_list: D000891:Anti-Infective Agents, Local; D002021:Buffers; D014867:Water; D014260:Triclosan