Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay

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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. Cytometry 36:340–348, 1999.