Abstract
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-α, 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 β-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 β-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.
Original language | English (US) |
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Pages (from-to) | 340-348 |
Number of pages | 9 |
Journal | Cytometry |
Volume | 36 |
Issue number | 4 |
DOIs | |
State | Published - Aug 1 1999 |
Externally published | Yes |
Keywords
- Annexin-V
- Degranulation
- Flow cytometry
- Mast cell
ASJC Scopus subject areas
- Pathology and Forensic Medicine
- Biophysics
- Hematology
- Endocrinology
- Cell Biology