Measuring Degranulation of Mast Cells

Robert J. Hohman1, Stephen C. Dreskin2

1 Oncor, Inc., Gaithersburg, Maryland, 2 University of Colorado Health Sciences Center, Denver, Colorado
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.26
DOI:  10.1002/0471142735.im0726s08
Online Posting Date:  May, 2001
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Abstract

This unit describes methods for measuring exocytosis of preformed mediators from secretory granules as an indication of IgE receptor‐mediated activation of mast cells. The first basic protocol describes the measurement of biogenic amines (serotonin and histamine) secreted by activated rodent mast cells (for serotonin) or rodent and human mast cells (for histamine). The second basic and alternate protocols detail techniques for measuring the release of β‐glucuronidase, an enzyme that is synthesized by human and rodent mast cells, stored in secretory granules, and released during degranulation. Methods for assaying other enzymes released during degranulation, such as β‐hexosaminidase and tryptase, are discussed in the . These protocols can also be applied to basophils where appropriate.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Serotonin or Histamine Secretion
  • Basic Protocol 2: Measurement of β‐Glucuronidase Secretion
  • Alternate Protocol 1: Microassay of β‐Glucuronidase Secretion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Measurement of Serotonin or Histamine Secretion

  Materials
  • Rat basophilic leukemia (RBL) cells
  • Eagle minimum essential medium containing 16% FCS (EMEM‐16)
  • Trypsin‐EDTA (GIBCO/BRL)
  • 3H‐labeled serotonin: 1 mCi/ml [3H]5′‐hydroxytryptamine binoxylate (15 to 30 Ci/mmol; Du Pont NEN #NET‐398; for measuring serotonin)
  • Mouse monoclonal anti‐DNP IgE (ICN Immunochemicals)
  • recipe1× sodium PIPES buffer
  • Dinitrophenyl–human albumin (DNP‐albumin; Sigma)
  • 1% (v/v) Triton X‐100 in phosphate‐buffered saline (PBS; appendix 2A)
  • Histamine radioimmunoassay (RIA) (Amac; for measuring histamine)
  • Biodegradable liquid scintillation cocktail
  • 75‐cm2 tissue culture flasks
  • 50‐ml polypropylene centrifuge tubes
  • Tabletop centrifuge and rotor (e.g., Sorvall RT‐6000B centrifuge with H‐1000B rotor)
  • 24‐well culture plates
  • Repeating pipettor: Eppendorf repeater pipet with sterile 12.5‐ml and nonsterile 0.5‐ml Combitip reservoirs ( Brinkmann)
  • Vacuum trap and vacuum manifold with four 13‐mm prongs (Thomas Scientific)
  • Liquid scintillation vials
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Measurement of β‐Glucuronidase Secretion

  Materials
  • recipe0.1 M sodium acetate buffer, pH 4.5
  • 0‐ to 50‐µg dilution of phenolphthalein standard solution (optional)
  • 0.01 M phenolphthalein glucuronic acid (Sigma; store at −4°C)
  • recipe0.4 M glycine stop solution, pH 10.5
  • 12 × 75–mm polypropylene test tubes
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Microassay of β‐Glucuronidase Secretion

  Additional Materials
  • 96‐well microtiter plates, nonsterile
  • Microtiter plate reader
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
   Beaven, M.A., Rogers, J., Moore, J.P., Hesketh, T.R., Smith, G.A., and Metcalfe, J.C. 1984. The mechanism of the calcium signal and correlation with histamine release in 2H3 cells. J. Biol. Chem. 259:7129‐7136.
   Dreskin, S.C., Probluda, V.S., and Metzger, H. 1989. IgE receptor‐mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells. J. Immunol. 142:4407‐4415.
   Fishman, W.H., Kato, K., Anstiss, C.L., and Green, S. 1967. Human serum β‐glucuronidase: Its measurement and some of its properties. Clin. Chim. Acta 15:435‐447.
   Glaser, J.H. and Sly, W.S. 1973. β‐Glucuronidase deficiency mucopolysaccharidosis: Methods for enzymatic diagnosis. J. Lab. Clin. Med. 82:969‐977.
   Lawrence, I.D., Warner, J.A., Cohan, V.L., Hubbard, W.C., Kagey‐Sobotka, A., and Lichtenstein, L.M. 1987. Purification and characterization of human skin mast cells. Evidence of human mast cell heterogeneity. J. Immunol. 139:3062‐3069.
   Mohr, F.C. and Fewtrell, C. 1987. The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. J. Biol. Chem. 262:10638‐10643.
   Schwartz, L.B., Lewis, R.A., Seldin, D., and Austen, K.F. 1981. Acid hydrolases and tryptase from secretory granules of dispersed human lung mast cells. J. Immunol. 126:1290‐1294.
   Schwartz, L.B. 1990. Tryptase, a mediator of human mast cells. J. Allergy Clin. Immunol. 86:594‐598.
   Siraganian, R.P. and Hook, W.A. 1986. Histamine release and assay methods for the study of human allergy. In Manual of Clinical Laboratory Immunology (N.R. Rose, H. Friedman, and J.C. Fahey, eds.) pp. 675‐684. American Society for Microbiology, Washington, D.C.
   Siraganian, R.P. and Hook, W.A. 1992. Histamine release and assay methods for the study of human allergy. In Manual of Clinical Laboratory Immunology (N.R. Rose, E.C. de Macario, J.C. Fahey, H. Friedman, and G.M. Penn, eds.) pp. 675‐684. American Society for Microbiology, Washington, D.C.
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