Isolation of Mast Cell Granules

Ken A. Lindstedt1, Petri T. Kovanen1

1 Wihuri Research Institute, Helsinki, Finland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.16
DOI:  10.1002/0471143030.cb0316s29
Online Posting Date:  January, 2006
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Abstract

The mast cell is a multipotent inflammatory cell that has been shown to participate in the pathogenesis of a variety of diseases, such as immediate hypersensitivity reactions, arthritis, atherosclerosis, and heart failure. Upon stimulation, mast cells exocytose cytoplasmic secretory granules into their extracellular microenvironment. These granules are modified lysosomes containing preformed mediators such as histamine, neutral proteases, cytokines, and growth factors embedded in a heparin proteoglycan matrix. When exposed to the extracellular fluid, the soluble components of the granules (e.g., histamine and cytokines) diffuse away, whereas the heparin proteoglycans and the mast cell–specific neutral proteases (e.g., chymase) remain tightly bound to each other, forming proteolytically active intra‐ and extracellular granule remnants. This unit describes a method to isolate rat serosa mast cell granule remnants in experiments aimed at determining the role of mast cell activation and degranulation in disease.

Keywords: Mast cells; granules; degranulation; granule remnants

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Rat Serosal Mast Cells
  • Basic Protocol 2: Isolation of Mast Cell Granules
  • Alternate Protocol 1: Isolation of Membrane‐Covered Granules
  • Alternate Protocol 2: Bulk Preparation of Mast Cell Granules
  • Support Protocol 1: Histamine Assay
  • Support Protocol 2: Quantification of Lactate Dehydrogenase Using the Cytotoxicity Detection Kit (Roche)
  • Support Protocol 3: Verification of Granule Morphology and Purity
  • Support Protocol 4: Determination of Heparin Proteoglycans
  • Support Protocol 5: Measurement of Chymase Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Rat Serosal Mast Cells

  Materials
  • Male Wistar rats, 300 to 500 g
  • CO 2 gas
  • Ethanol
  • Buffer A (see recipe)
  • Heparin (Sigma)
  • Medium A (see recipe)
  • PBS with magnesium and calcium (GIBCO)
  • Moore & James staining solution (see recipe)
  • Glass tank with cover
  • Sterilized scissors and forceps (surgical and anatomical)
  • 20‐ and 10‐ml syringes
  • 10‐ml disposable pipets
  • 18‐G needles
  • 15‐ and 50‐ml centrifuge tubes
  • 56°C water bath
  • Centrifuge (e.g., Eppendorf 5810R)
  • 150 × 15–mm petri dishes
  • 37°C, 5% CO 2 humidified incubator
  • Hemacytometer
NOTE: Treat the mast cells gently throughout the protocol to avoid spontaneous degranulation and loss of granules.

Basic Protocol 2: Isolation of Mast Cell Granules

  Materials
  • Mast cells (see protocol 1)
  • PBS with magnesium and calcium (GIBCO), cold and room temperature
  • Compound 48/80 (Sigma)
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge
  • 37°C, 5% CO 2 humidified incubator

Alternate Protocol 1: Isolation of Membrane‐Covered Granules

  Materials
  • Purified mast cells (see protocol 1)
  • Buffer B (see recipe)
  • 0.1% (v/v) Triton X‐100 in buffer B (see recipe for buffer B)
  • PBS with magnesium and calcium (GIBCO)
  • 1.5‐ml microcentrifuge tubes
  • Sonicator (e.g., Model Pul 125, Kerry Ultrasonics)

Alternate Protocol 2: Bulk Preparation of Mast Cell Granules

  Materials
  • Purified mast cells (see protocol 1)
  • 0.3 M sucrose
  • Liquid nitrogen
  • Buffer A (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 37°C water bath
  • Additional reagents and equipment for determination of protein concentration by the method of Lowry ( appendix 3H)

Support Protocol 1: Histamine Assay

  Materials
  • Activated mast cells (see protocol 2)
  • Trichloroacetic acid (TCA)
  • Orthopthalaldehyde (OPTA, Fluka)
  • Methanol
  • Histamine (Sigma)
  • 0.01, 0.05, and 2 N H 2SO 4
  • 1 N NaOH
  • 1.5‐ml microcentrifuge tubes
  • Fluorometer (340‐nm excitation and 443‐nm emission filters)

Support Protocol 2: Quantification of Lactate Dehydrogenase Using the Cytotoxicity Detection Kit (Roche)

  Materials
  • Cell‐free supernatant from mast cells (see protocol 2; 1×106 activated mast cells)
  • PBS
  • Cytotoxicity detection kit (Roche) containing:
    • Catalyst: diaphorase/NAD+ mixture and lyophilizate, stabilized
    • Dye solution: iodotetrazolium chloride (INT) and sodium lactate
  • 96‐well microtiter plates with flat‐bottomed wells
  • Microtiter plate reader (490 nm)

Support Protocol 3: Verification of Granule Morphology and Purity

  Materials
  • Mast cell granules (see protocol 2)
  • 2.5% (v/v) glutaraldehyde/100 mM sodium phosphate buffer, pH 7.2
  • 1% (w/v) osmium tetroxide/100 mM sodium phosphate buffer, pH 7.2
  • 50%, 70%, 94%, and 99% ethanol
  • LX‐112 embedding medium (Ladd Research)
  • Uranyl acetate (Ultrostain I, Leica)
  • Lead citrate (Ultrostain II, Leica)
  • Ultramicrotome
  • Automated Leica EMStain apparatus (Leica)
  • Electron microscope

Support Protocol 4: Determination of Heparin Proteoglycans

  Materials
  • Sepraphore III cellulose polyacetate electrophoresis strips (2.5 × 15.2–cm, Pall Corporation)
  • Heparin standards (Sigma)
  • Mast cell granule remnants (see protocol 2)
  • 0.2% (w/v) Alcian blue staining solution (see recipe)
  • Destaining solution (see recipe)
  • DMSO
  • 10‐ml disposable plastic tubes (DMSO‐resistant)
  • Platform shaker
  • Whatman no. 3 filter paper
  • Spectrophotometer

Support Protocol 5: Measurement of Chymase Activity

  Materials
  • Isolated mast cell granules (see protocol 2)
  • 0.5 mM n‐benzoyl‐L‐tyrosine ethyl ester (BTEE; Sigma) in 100 mM NaCl/50 mM Tris⋅Cl, pH 7.4
  • Spectrophotometer (256 nm)
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Figures

Videos

Literature Cited

Literature Cited
   Aridor, M., Traub, L.M., and Sagi‐Eisenberg, R. 1990. Exocytosis in mast cells by basic secretagogues: Evidence for direct activation of GTP‐binding proteins. J. Cell Biol. 111:909‐917.
   Bartold, P.M. and Page, R.C. 1985. A microdetermination method for assaying glycosaminoglycans and proteoglycans. Anal. Biochem. 150:320‐324.
   Bergendorff, A. and Uvnäs, B. 1972. Storage of 5‐hydroxytryptamine in rat mast cells. Evidence for an ionic binding to carboxyl groups in a granule heparin‐protein complex. Acta Physiol. Scand. 84:320‐331.
   Kokkonen, J.O. and Kovanen, P.T. 1985. Low density lipoprotein degradation by rat mast cells: Demonstration of extracellular proteolysis caused by mast cell granules. J. Biol. Chem. 260:14756‐14763.
   Kokkonen, J.O., Lindstedt, K.A., and Kovanen, P.T. 1995. Role of mast cell proteases and proteoglycans in lipoprotein metabolism. In Mast Cell Proteases in Immunology and Biology. Clinical Allergy and Immunology. (G.H. Caughey, ed.) pp. 257‐287. Marcel Dekker, Inc. New York.
   Kovanen, P.T. 1993. The mast cell—A potential link between inflammation and cellular cholesterol deposition in atherogenesis. Eur. Heart J. 14:105‐117.
   Krüger, P.G., Lagunoff, D., and Wan, H. 1980. Isolation of rat mast cell granules with intact membranes. Exp. Cell. Res. 129:83‐93.
   Lindstedt, K.A., Kokkonen, J.O., and Kovanen, P.T. 1992. Soluble heparin proteoglycans release from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor‐mediated phagocytosis. J. Lipid Res. 33:65‐75.
   Thon, I.‐L. and Uvnäs, B. 1966. Mode of storage of histamine in mast cells. Acta Physiol. Scand. 67:455‐470.
   Thon, I.‐L. and Uvnäs, B. 1967. Degranulation and histamine release, two consecutive steps in the response of rat mast cells to compound 48/80. Acta Physiol. Scand. 71:303‐315.
   Woodbury, R.G., Everitt, M.T., and Neurath, H. 1981. Mast cell proteases. Methods Enzymol. 80:588‐609.
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