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Beschreibung

Introduction Degranulation, the secretion of cytoplasmic granules, is a key step in the inflammatory response of leukocytes (e.g. basophils, neutrophils, eosinophils, and mast cells). In addition to histamine, these secretory granules contain many proinflammatory mediators including heparin, cytokines, chemokines, and many proteases (Hallgren, 2001). Tryptase, a tetrameric serine proteinase, has emerged as the major component of mast cell granules, comprising up to 20% of the total protein of mast cells derived from lung, colon and skin tissue (He, 2004, He, 1998, Schwartz, 1986). Because it is stored almost exclusively in mast cells, tryptase is a popular indicator of mast cell activation and a target for therapeutic intervention in allergic diseases. The Chemicon Mast Cell Degranulation Assay Kit provides a quick, efficient and sensitive system for evaluation of tryptase activity in cell lysates, supernatants or for inhibitor screening. Test Principle Chemicon's Mast Cell Degranulation Assay Kit provides a simple and convenient means for assaying tryptase activity. The assay is based on spectophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate tosyl-gly-pro-lys-pNA. The free pNA can then be quantified using a spectrophotometer or a microtiter plate reader at 405 nm. A pNA Standard is provided as a comparison control. Calcium Ionophore A23187 (Calcimycin) is included in the kit for degranulation induction of samples. Calcimycin has been a well-documented inducer of tryptase release from human mast cells (2,3). A tryptase inhibitor, protamine, is included as a test inhibitor for screening purposes. Protamine is a potent heparin antagonist that competitively binds heparin, an essential stabilizing factor of mast cell tryptase (Hallgren, 2001, Schwartz, 1986). Protamine has been shown to inhibit both human and mouse tryptase (Hallgren, 2001). The kit's Assay Buffer contains heparin for tryptase stabilization.