Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay
✍ Scribed by S.D. Demo; E. Masuda; A.B. Rossi; B.T. Throndset; A.L. Gerard; E.H. Chan; R.J. Armstrong; B.P. Fox; J.B. Lorens; D.G. Payan; R.H. Scheller; J.M. Fisher
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 323 KB
- Volume
- 36
- Category
- Article
- ISSN
- 0196-4763
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✦ Synopsis
Background: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-␣, and leukotrienes. Methods: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme -hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. Results: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and -hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein.
Conclusions:
The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis. Cytometry 36: