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Measurement of Exocytosis in Genetically Manipulated Mast Cells

  • Ofir Klein
  • Nurit P. Azouz
  • Ronit Sagi-EisenbergEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2233)

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.

Key words

Mast cells Regulated exocytosis NPY-mRFP Transfection Microscopy 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Ofir Klein
    • 1
    • 2
  • Nurit P. Azouz
    • 1
    • 3
  • Ronit Sagi-Eisenberg
    • 1
    Email author
  1. 1.Department of Cell and Developmental Biology, Sackler Faculty of MedicineTel Aviv UniversityTel AvivIsrael
  2. 2.Department of Molecular GeneticsWeizmann Institute of ScienceRehovotIsrael
  3. 3.Department of Pediatrics, Cincinnati Children Hospital Medical CenterUniversity of Cincinnati College of MedicineCincinnatiUSA

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