Assays for Regulated Exocytosis of Mast Cell Granules

Abstract

Mast cells are important effectors in innate and adaptive immune responses. They contain numerous secretory granules filled with inflammatory mediators in their cytoplasm. Exocytosis of granular content does not take place until the cell receives an appropriate stimulus such as the aggregation of IgE antibody bound to high‐affinity IgE receptors by specific antigen. This process is therefore referred to as regulated exocytosis. A characteristic of mast cell exocytosis is that it does not involve release of a few individual granules, but rather, a large fraction of the granular content is released due to compound exocytosis, implicating the occurrence of granule‐to‐granule and granule‐to‐plasma membrane fusion. This unit describes assays that measure the release of granular content from mast cells. They include in vitro colorimetric‐, radiolabel‐, or antibody detection–based assays for substances stored in the granules. Given that animal models for basophil and mast cell activation are being used with increasing frequency, the unit also includes protocols to measure exocytosis of mast cell granule content in vivo.

Ikaros limits basophil development by suppressing C/EBP-α expression

“…32 Briefly, bone marrow-derived mast cells were sensitized with 0.5 mg/mL anti-dinitrophenol (DNP) IgE (Sigma-Aldrich, St. Louis, MO) for 3 hours in RPMI 1640 with IL-3 and SCF. Cells were stimulated with DNP-human serum albumin (Sigma-Aldrich) for 30 minutes in Tyrode's buffer before supernatant collection and lysis of the cell pellet with 0.5% Triton X-100.…”

p66Shc Is a Negative Regulator of FcεRI-Dependent Signaling in Mast Cells

“…A total of 0.2 3 10 6 cells/sample was left untreated or sensitized with anti-DNP IgE (1 mg/ml) overnight at 37˚C, washed, and challenged for 20 min with DNP-ALB (20-2000 ng/ml) or treated with A23187 (500 ng/ml), ATP (3 mM), and thapsigargin (2 mM) (Sigma).The b-hexosaminidase content of supernatants and cells was determined, as described (23), and calculated as follows: b-hexosaminidase released/ (b-hexosaminidase in supernatants + b-hexosaminidase in cells) 3100. Alternatively, measurement of degranulation by Annexin V binding was performed on peritoneal cells, as described (24). Briefly, 1 3 10 6 peritoneal cells cultured in WEHI-3B-conditioned medium were sensitized with anti-DNP IgE (1 mg/ml) for 3 h at room temperature, washed, and challenged with DNP-ALB (20 ng/ml) for 30 s. The reaction was stopped by adding ice-cold Annexin V-binding buffer and transferring the tube on ice.…”

A Heterobivalent Ligand Inhibits Mast Cell Degranulation via Selective Inhibition of Allergen–IgE Interactions In Vivo

“…Cross-linking of the IgE antibodies initiates an intracellular signaling cascade resulting in degranulation, which causes the release of preformed mediators stored in cytoplasmic granules including vasoactive amines, neutral proteases, proteoglycans, cytokines, and chemokines (4). Current therapies for allergic responses include epinephrine, anti-histamines, steroids, and IgE-depleting therapies such as omalizumab.…”