IMM001 | Mast Cell Degranulation Assay Kit

IMM001
96 assays  
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      Overview

      Replacement Information

      Key Spec Table

      Key Applications
      ACT
      Description
      Catalogue NumberIMM001
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionMast Cell Degranulation Assay Kit
      OverviewIntroduction

      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.
      Materials Required but Not Delivered· Microcentrifuge and 1.5 mL microcentrifuge tubes.

      · 37°C water bath or Incubator.

      · Spectrophotometer or microplate reader.

      · Tissue culture treated or non-tissue culture treated plate.

      · Adjustable volume pipettor with disposable tips.

      · Pulse sonicator.

      · 96-well microtiter plate

      · 1X PBS or HBSS

      · DMSO
      References
      Product Information
      Components
      • Tryptase Positive Control (Part No. 2003650): 20 μL of 0.1 mg/mL solution.
      • 5X Assay Buffer (Part No. 2003649): 20 mL containing 0.1% heparin.
      • Calcium Ionophore A23187 (Part No. 2003652): 100 μL of 1 mM in DMSO.
      • Tryptase Inhibitor (Protamine) (Part No. 2003651): 250 μL of 10 mM solution.
      • Tryptase Substrate (tosyl-gly-pro-lys-pNA) (Part No. 90569): 3.125 mg.
      • pNA Standard (Part No. 90085): 250 μL of 10 mM in DMSO.
      Applications
      ApplicationThe Mast Cell Degranulation Assay Kit provides a quick, efficient & sensitive system for evaluation of tryptase activity in culture supernatants, cell lysates or other tryptase-containing samples.
      Key Applications
      • Activity Assay
      Application NotesThe Mast Cell Degranulation Assay Kit provides a quick, efficient and sensitive system for evaluation of tryptase activity in culture supernatants, cell lysates or other tryptase-containing samples. Testing of purified tryptase enzyme, in vitro inhibitor screening and the study of cell degranulation can also be performed with this assay.

      The Chemicon Mast Cell Degranulation Assay Kit is intended for research use only, and not for diagnostic or therapeutic applications.
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage ConditionsStore kit materials (as provided) at -20°C up to their expiration date. See Storage of Reagents for component specific instructions.

      Storage of Reagents

      1. Tryptase Positive Control: Thaw vial at 2-8°C. Aliquot and store at -20°C up to the vial's expiration date. Avoid multiple freeze/thaw cycles.

      2. 5X Assay Buffer: Dilute the 5X Assay Buffer with deionized water. Stir to homogeneity and store at 2-8°C up to its expiration date.

      3. Calcium Ionophore A23187: Thaw vial at 2-8°C. Aliquot and store at -20°C up to the vial's expiration date. Avoid multiple freeze/thaw cycles.

      4. Tryptase Inhibitor: Thaw vial at 2-8°C. Aliquot and store at -20°C up to the vial's expiration date. Avoid multiple freeze/thaw cycles.

      5. Tryptase Substrate: Reconstitute with 2 mL of 1X Assay Buffer (2.5 mM stock solution). Aliquot and store at -20°C up to 3 months. Avoid multiple freeze/thaw cycles.

      6. pNA Standard: Thaw vial at 2-8°C. Aliquot and store at -20°C up to the vial's expiration date. Avoid multiple freeze/thaw cycles.

      Note: Positive Control, Substrate, pNA Standard, and Calcium Ionophore are light sensitive; amber vials or equivalent should be used. Keep materials away from light as much as possible and during storage.
      Packaging Information
      Material Size96 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      SDS

      Title

      Safety Data Sheet (SDS) 

      References

      Reference overviewPub Med ID
      Mast cell activation and degranulation promotes renal fibrosis in experimental unilateral ureteric obstruction.
      Shaun A Summers,Poh-Yi Gan,Lakshi Dewage,Frank T Ma,Joshua D Ooi,Kim M O'Sullivan,David J Nikolic-Paterson,A Richard Kitching,Stephen R Holdsworth
      Kidney international  82  2012

      Show Abstract
      22673890 22673890

      Technical Info

      Title
      96 well Template 2 Up
      96 well Template Single

      Data Sheet

      Title
      Mast Cell Degranulation Assay Kit - Data Sheet

      User Guides

      Title
      Mast Cell Degranulation Assay Kit