Measuring Mast Cell Mediator Release

Hye Sun Kuehn1, Madeleine Radinger1, Alasdair M. Gilfillan1

1 Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.38
DOI:  10.1002/0471142735.im0738s91
Online Posting Date:  November, 2010
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Abstract

Mediators released from activated mast cells are responsible for the allergic inflammatory reactions associated with disease states such as anaphylaxis and atopy. These mediators are released as a consequence of immediate degranulation and phospholipid metabolism upon mast cell activation, followed by delayed cytokine gene expression. Thus, techniques that monitor indices of these events in mast cell culture systems, in association with biochemical analysis of parameters of cell signaling, are critical to our understanding of the molecular mechanisms regulating mast cell–mediated disease. Furthermore, these systems can be adapted for high‐throughput screens to identify potential inhibitors of mast cell activation that may provide potential leads for novel therapies for these diseases. In this unit, we describe approaches that can be readily used or adapted to a variety of rodent and human mast cell culture systems for the determination of degranulation, phospholipid‐derived inflammatory mediator production, and cytokine generation. Curr. Protoc. Immunol. 91:7.38.1‐7.38.9. © 2010 by John Wiley & Sons, Inc.

Keywords: cytokines; degranulation; eicosanoids; FcɛRI; human mast cells (HuMCs); KIT; mouse bone marrow–derived mast cells (BMMCs); stem cell factor (SCF)

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Mast Cell Degranulation as Monitored by β‐Hexosaminidase Release
  • Basic Protocol 2: Measurement of Eicosanoid (PGD2, LTC4) Generation in Mast Cells
  • Basic Protocol 3: Measurement of Cytokine Release from Mast Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Mast Cell Degranulation as Monitored by β‐Hexosaminidase Release

  Materials
  • C57BL/6J mice (The Jackson Laboratory) for obtaining BMMC
  • BMMC‐MC/9 medium (unit 3.23)
  • CD34+‐peripheral blood progenitor cells or human LAD2 mast cell line (see unit 7.37) for obtaining HuMC
  • Human mast cell culture medium or LAD2 cell culture medium (unit 7.37)
  • Dinitrophenyl‐human serum albumin conjugate (DNP‐HSA) (Sigma‐Aldrich)
  • Human myeloma IgE (Calbiochem), biotinylated using NHS‐biotin (Zymed); see unit 5.3, Support Protocol 2
  • Mouse monoclonal anti‐DNP‐IgE (clone SPE‐7; Sigma‐Aldrich)
  • HEPES buffer, pH 7.4 (see recipe)
  • Stimulants [e.g., dinitrophenyl–human serum albumin conjugate (DNP‐HSA; Sigma‐Aldrich) for BMMC or streptavidin for HuMC] or inhibitors of interest
  • p‐nitrophenyl N‐acetyl‐β‐D‐glucosamide (PNAG; Sigma‐Aldrich)
  • Citrate buffer, pH 4.5: 40 mM citric acid/20 mM Na 2HPO 4⋅7 H 2O
  • 0.1% (v/v) Triton X‐100
  • 400 mM glycine, pH 10.7
  • Centrifuge: e.g., Sorvall RT‐6000B refrigerated centrifuge
  • 96‐well plates
  • Heating block or warm air oven
  • Spectrophotometer with microtiter plate reader
  • Additional reagents and equipment for isolation and culture of mouse mast cells (unit 3.23) and human mast cells (unit 7.37)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: All solutions are made in distilled water. All solutions and media must be filtered and kept at 4°C.

Basic Protocol 2: Measurement of Eicosanoid (PGD2, LTC4) Generation in Mast Cells

  Materials
  • Prostaglandin D 2‐MOX EIA kit (e.g., Cayman Chemical)
  • Leukotriene C 4 EIA kit (e.g., Cayman Chemical)
  • 96‐well plate or 1.5‐ml polypropylene microcentrifuge tubes
  • Additional reagents and equipment for measuring mast cell degranulation as monitored by β‐hexosaminidase release ( protocol 1)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: Prepare ultrapure water for the eicosanoid measurement assay. Water must be deionized and free of trace organic contaminations. To accomplish the latter, use an activated carbon filter cartridge or organic scavengers.

Basic Protocol 3: Measurement of Cytokine Release from Mast Cells

  Materials
  • Cytokine assay kit (e.g., R&D systems)
  • 24‐ or 48‐well plate
  • Additional reagents and equipment for measuring mast cell degranulation as monitored by β‐hexosaminidase release ( protocol 1)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Literature Cited

Literature Cited
   Blank, U., and Rivera, J. 2006. Assays for regulated exocytosis of mast cell granules. Curr. Protoc. Cell Biol. 32:15.11.1‐15.11.18.
   Boyce, J.A. 2007. Mast cells and eicosanoid mediators: A system of reciprocal paracrine and autocrine regulation. Immunol. Rev. 217:168‐185.
   Brown, J.M., Wilson, T.M., and Metcalfe, D.D. 2008. The mast cell and allergic diseases: Role in pathogenesis and implications for therapy. Clin. Exp. Allergy 38:4‐18.
   Gilfillan, A.M. and Tkaczyk, C. 2006. Integrated signalling pathways for mast‐cell activation. Nat. Rev. Immunol. 6:218‐230.
   Hundley, T.R., Gilfillan, A.M., Tkaczyk, C., Andrade, M.V., Metcalfe, D.D., and Beaven, M.A. 2004. Kit and FcepsilonRI mediate unique and convergent signals for release of inflammatory mediators from human mast cells. Blood 104:2410‐2417.
   Kuehn, H.S. and Gilfillan, A.M. 2007. G protein‐coupled receptors and the modification of FcepsilonRI‐mediated mast cell activation. Immunol. Lett. 113:59‐69.
   Marshall, J.S. 2004. Mast‐cell responses to pathogens. Nat. Rev. Immunol. 4:787‐799.
   Metcalfe, D.D., Baram, D., and Mekori, Y.A. 1997. Mast cells. Physiol. Rev. 77:1033‐1079.
   Qiao, H., Andrade, M.V., Lisboa, F.A., Morgan, K., and Beaven, M.A. 2006. FcɛR1 and toll‐like receptors mediate synergistic signals to markedly augment production of inflammatory cytokines in murine mast cells. Blood 107:610‐618.
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