Measurement of Exocytosis in Genetically Manipulated Mast Cells.

Abstract

The hallmark of mast cell activation is secretion of immune mediators by regulated exocytosis. Measurements of mediator secretion from mast cells that are genetically manipulated by transient transfections provide a powerful tool for deciphering the underlying mechanisms of mast cell exocytosis. However, common methods to study regulated exocytosis in bulk culture of mast cells suffer from the drawback of high signal-to-noise ratio because of their failure to distinguish between the different mast cell populations, that is, genetically modified mast cells versus their non-transfected counterparts. In particular, the low transfection efficiency of mast cells poses a significant limitation on the use of conventional methodologies. To overcome this hurdle, we developed a method, which discriminates and allows detection of regulated exocytosis of transfected cells based on the secretion of a fluorescent secretory reporter. We used a plasmid encoding for Neuropeptide Y (NPY) fused to a monomeric red fluorescent protein (NPY-mRFP), yielding a fluorescent secretory granule-targeted reporter that is co-transfected with a plasmid encoding a gene of interest. Upon cell trigger, NPY-mRFP is released from the cells by regulated exocytosis, alongside the endogenous mediators. Therefore, using NPY-mRFP as a reporter for mast cell exocytosis allows either quantitative, via a fluorimeter assay, or qualitative analysis, via confocal microscopy, of the genetically manipulated mast cells. Moreover, this method may be easily modified to accommodate studies of regulated exocytosis in any other type of cell.