Real-time imaging of mast cell degranulation in vitro and in vivo

Biochem Biophys Res Commun. 2016 Oct 21;479(3):517-522. doi: 10.1016/j.bbrc.2016.09.100. Epub 2016 Sep 21.

Abstract

Mast cells undergo degranulation in response to various stimuli and rapidly release pre-formed mediators present in secretory granules, leading to immediate-type allergic reactions. Mast cell degranulation is commonly detected and quantified in vitro by measuring histamine or β-hexosaminidase released to culture medium. However, this type of assay cannot monitor degranulation of individual cells in real time, and it is not suitable for in vivo detection of degranulation. At the aim of real time imaging of mast cell degranulation at single cell level, we here developed a fluorescent protein-based indicator of degranulation, designated immuno-pHluorin (impH). When expressed in mast cells, impH is located in the membrane of secretory granules and non-fluorescent under homeostatic conditions while it turns fluorescent following degranulation, due to the pH change inside of granules during exocytosis. impH enabled us to detect polarized degranulation within one single cell when mast cells were stimulated via the small area of cell surface. Transplantation of impH-expressing mast cells into mast cell-deficient mice demonstrated that impH could function as a real-time indicator of degranulation in vivo. Thus, impH is a useful tool for imaging of mast cell activation and degranulation in vitro and in vivo, and may be applied for screening of reagents regulating mast cell degranulation.

Keywords: Degranulation; Exocytosis; Imaging; Mast cell; VAMP-8; pH sensor.

MeSH terms

  • Animals
  • Bone Marrow Cells / cytology
  • Cell Degranulation*
  • Culture Media
  • Exocytosis
  • Fluorescent Dyes / chemistry
  • Green Fluorescent Proteins / chemistry*
  • Histamine / chemistry
  • Histamine Release*
  • Homeostasis
  • Hydrogen-Ion Concentration
  • Luminescent Proteins / chemistry
  • Mast Cells / cytology*
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • R-SNARE Proteins / chemistry
  • Secretory Vesicles / metabolism
  • Time Factors
  • beta-N-Acetylhexosaminidases / chemistry

Substances

  • Culture Media
  • Fluorescent Dyes
  • Luminescent Proteins
  • PHluorin
  • R-SNARE Proteins
  • VAMP8 protein, human
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Histamine
  • beta-N-Acetylhexosaminidases