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

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-␣, 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.

Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence

“…2 and Supplementary Movie 2), indicating that the observed phenomenon does not depend on the stimulus strength. We also employed an Annexin-V-binding assay to monitor hMC degranulation following anti-IgE stimulation 35 . The results obtained using this method corroborated the results observed using avidin ( Supplementary Fig.…”

“…A total of 5 Â 10 4 hMCs previously sensitized with human IgE (1 mg ml À 1 ) were plated on Poly-D-Lysine (Sigma)-coated Lab-Tek chambered coverglass (Nunc) in Tyrode's buffer supplemented with 8 mg ml À 1 avidin-sulforhodamine 101 (Av.SRho, highly cationic glycoprotein that selectively stains mast cell granules 34 ) or Annexin-V-Alexa647 (final 3% v/v) 35 as indicated and warmed at 37°C for 20 min. Cells were stimulated with 2.5 mg ml À 1 goat anti-human IgE at time ¼ 0.…”

“…A total of 5 Â 10 4 hMCs previously sensitized or not with human anti-CD20 IgE (1 mg ml À 1 ) were plated on Poly-D-Lysine (Sigma)-coated Lab-Tek chambered coverglass (Nunc) in Tyrode's buffer supplemented with 8 mg ml À 1 avidin-sulforhodamine 101 (Av.SRho, highly cationic glycoprotein that selectively stains mast cell granules 34 , Sigma) or Annexin-V-Alexa647 (final 3% v/v) 35 Confocal microscopy analysis of JY cell/hMC conjugates. A total of 2.5 Â 10 4 hMCs sensitized or not with human anti-CD20 IgE (1 mg ml À 1 ) were plated on Poly-D-Lysine-coated diagnostic slides (Thermo Scientific) in 25 ml Tyrode's buffer supplemented with 8 mg ml À 1 Av.SRho for 20 min at 37°C.…”

Dnmt3a restrains mast cell inflammatory responses

“…We therefore assessed mast cell responses to physiological stimulation with IgE and antigen. Interestingly, upon acute stimulation, mast cells lacking Dnmt3a showed significantly increased ability to release the content of their cytoplasmic granules, as assessed both by annexin V binding [which occurs at sites of secretory granule fusion with the plasma membrane (19)(20)(21)] and release of β-hexosaminidase (an enzyme normally stored in cytoplasmic granules) in the culture supernatant ( Fig. 1 D and E).…”

Tubulin Polymerization-promoting Protein (TPPP/p25α) Promotes Unconventional Secretion of α-Synuclein through Exophagy by Impairing Autophagosome-Lysosome Fusion

“…Characteristics-Exocytosis of late endosomal elements, including amphisomes, which arise from fusion with late endosomes, causes insertion of phosphatidylserine into the exoplasmic leaflet of the plasma membrane, and this can be detected by annexin-V staining (41). As binding of annexin-V to the cell surface is also used for detection of apoptosis, we co-stained the cells with anticaspase-3 antibodies to separate the two phenomena.…”