A convenient fluorimetry-based degranulation assay using RBL-2H3 cells

Type I hypersensitivity is triggered by mast cell degranulation, a stimulus-induced exocytosis of preformed secretory granules (SGs) containing various inflammatory mediators. The degree of degranulation is generally expressed as a percentage of secretory granule markers (such as beta-hexosaminidase and histamine) released into the external solution, and considerable time and labor are required for the quantification of markers in both the supernatants and cell lysates. In this study, we developed a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells. During degranulation, the styryl dye FM1-43 in the external solution fluorescently labeled the newly exocytosed SGs, whose increase in intensity was successively measured using a fluorescence microplate reader. In addition to the rate of beta-hexosaminidase secretion, the cellular FM1-43 intensity successfully represented the degree and kinetics of degranulation under various conditions, suggesting that this method facilitates multi-sample and/or multi-time-point analyses required for screening substances regulating mast cell degranulation. Graphical Abstract Extracellular FM1-43 progressively labeled rat basophilic leukemia (RBL-2H3) cells in a secretagogue-dependent manner, whose intensity represented the degree of degranulation.

Automated summary

In this study, a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells was developed. The assay successfully represented the degree and kinetics of degranulation, providing a tool for screening substances regulating mast cell degranulation.