Assays for Regulated Exocytosis of Mast Cell Granules

Ulrich Blank1, Juan Rivera2

1 Bichat Medical School, Paris, 2 National Insitutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.11
DOI:  10.1002/0471143030.cb1511s32
Online Posting Date:  October, 2006
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Abstract

Mast cells are important effectors in innate and adaptive immune responses. They contain numerous secretory granules filled with inflammatory mediators in their cytoplasm. Exocytosis of granular content does not take place until the cell receives an appropriate stimulus such as the aggregation of IgE antibody bound to high‐affinity IgE receptors by specific antigen. This process is therefore referred to as regulated exocytosis. A characteristic of mast cell exocytosis is that it does not involve release of a few individual granules, but rather, a large fraction of the granular content is released due to compound exocytosis, implicating the occurrence of granule‐to‐granule and granule‐to‐plasma membrane fusion. This unit describes assays that measure the release of granular content from mast cells. They include in vitro colorimetric‐, radiolabel‐, or antibody detection–based assays for substances stored in the granules. Given that animal models for basophil and mast cell activation are being used with increasing frequency, the unit also includes protocols to measure exocytosis of mast cell granule content in vivo.

Keywords: mast cell; regulated exocytosis; secretory granules; allergy; anaphylaxis

     
 
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Table of Contents

  • Detection of Soluble Mediators
  • Basic Protocol 1: Measurement of Histamine Release
  • Alternate Protocol 1: Measurement of Radiolabeled Serotonin Release
  • Alternate Protocol 2: Measurement of β‐Hexosaminidase Release
  • Basic Protocol 2: Measurement of Regulated Exocytosis by Annexin V Binding
  • Support Protocol 1: Bone Marrow Extraction for Preparation of Bone Marrow‐Derived Cultured Mast Cell (BMMC)
  • Basic Protocol 3: Measurement of Regulated Exocytosis Using the Passive Systemic Anaphylaxis (PSA) Method
  • Alternate Protocol 3: Detection of Regulated Exocytosis by Passive Systemic Anaphylaxis
  • Alternate Protocol 4: Detection of Regulated Exocytosis by Active Systemic Anaphylaxis
  • Alternate Protocol 5: Detecting Regulated Exocytosis by Passive Cutaneous Anaphylaxis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Measurement of Histamine Release

  Materials
  • RBL‐2H3 (ATCC no. CRL‐2256) or bone‐marrow mast cells (BMMC; see protocol 5), in late log phase (3 to 4 days after feeding)
  • Tyrode's buffer (see recipe)
  • 5‐[1,2‐3H(N)]‐hydroxytryptamine binoxalate (sp. act., 28.5 Ci/mmol; ViTrax Radiochemicals; http://www.vitrax.com/)
  • DNP‐specific murine IgE (Sigma‐Aldrich) or TNP‐specific murine IgE (BD PharMingen)
  • Antigen: dinitrophenol conjugated to human serum albumin (DNP 36‐HSA; Sigma‐Aldrich), trinitrophenol conjugated to ovalbumin (TNP‐OVA; Biosearch Technologies), or trinitrophenol conjugated to bovine serum albumin (TNP‐BSA; Biosearch Technologies)
  • Tyrode's buffer–E (see recipe)
  • 1% (v/v) Triton X‐100
  • Scintillation fluid, water soluble
  • Centrifuge
  • Scintillation vials

Alternate Protocol 1: Measurement of Radiolabeled Serotonin Release

  Materials
  • RBL‐2H3 cells (ATCC no. CRL‐2256) or bone‐marrow mast cells (BMMC; see protocol 5), in late log phase (3 to 4 days after feeding)
  • Culture medium for RBL‐2H3 cells (see recipe) or culture medium for BMMC (see recipe; if BMMC are used)
  • DNP‐specific murine IgE (Sigma‐Aldrich)
  • Tyrode's buffer (see recipe), room temperature and 37°C
  • Antigen: dinitrophenol conjugated to human serum albumin (DNP 36‐HSA; Sigma‐Aldrich)
  • 0.5% (v/v) Triton X‐100
  • pNAG substrate (see recipe)
  • Carbonate buffer (see recipe)
  • Culture medium for RBL‐2H3 cells (see recipe) without IL‐3
  • Flat‐bottom 96‐well microtiter plates
  • Refrigerated centrifuge with microtiter plate carrier
  • Microtiter plate reader
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Measurement of β‐Hexosaminidase Release

  Materials
  • RBL‐2H3 (ATCC no. CRL‐2256) or bone‐marrow mast cells (BMMC; see protocol 5), in late log phase (3 to 4 days after feeding)
  • Culture medium for RBL‐2H3 cells (see recipe) or culture medium for BMMC (see recipe; if BMMC are used)
  • 1 M HEPES, pH 7.3
  • DNP‐ or TNP‐specific murine IgE (Sigma‐Aldrich)
  • Tyrode's buffer (see recipe)
  • Antigen: DNP‐HSA or TNP‐HSA (Sigma‐Aldrich)
  • 10× annexin V binding buffer (see recipe), ice cold
  • Biotinylated annexin V (Invitrogen)
  • 0.1 mg/ml streptavidin‐phycoerythrin (streptavidin‐PE; Invitrogen) or streptavidin‐allophycocyanin (streptavidin‐APC; Invitrogen)
  • 50‐ml conical centrifuge tubes
  • Platform shaker
  • 12 × 15–mm round bottom FACS analysis test tubes (Becton Dickinson)
  • Fluorescence activated cell sorter (FACS; Becton Dickinson)

Basic Protocol 2: Measurement of Regulated Exocytosis by Annexin V Binding

  Materials
  • Mice, 8‐ to 12‐weeks old
  • 70% ethanol
  • Culture medium for BMMC (see recipe)
  • Culture medium for BMMC (see recipe) without IL‐3 (but containing SCF)
  • Culture medium for BMMC (see recipe) without IL‐3 or SCF
  • Dissection instruments
  • 3‐cc syringe with 30‐G needle
  • Centrifuge
  • 75‐cm2 tissue culture flasks
  • Additional reagents and equipment for euthanasia of mice (Donovan and Brown, )

Support Protocol 1: Bone Marrow Extraction for Preparation of Bone Marrow‐Derived Cultured Mast Cell (BMMC)

  Materials
  • Mice
  • 15 µg/ml IgE anti‐DNP antibody (Sigma‐Aldrich) in sterile PBS ( appendix 2A)
  • 2.5 mg/ml dinitrophenol conjugated to human serum albumin (DNP‐HSA; Sigma‐Aldrich) in sterile PBS ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Histamine enzyme immunoassay kit (Beckman Coulter) containing:
    • Anti–histamine antibody–coated microtiter plate
    • Zero standard
    • Histamine standards
    • Control
    • Histamine–alkaline phosphatase conjugate
    • Conjugate diluent H
    • Acylation reagent
    • Release buffer
    • 20× wash solution
    • Substrate buffer (diethylamine)
    • p‐nitrophenylphosphate substrate tablets
    • Stop solution (NaOH)
  • Gauze pads, sterile
  • Mouse cages
  • Heat lamp
  • 1‐ml syringes with 26‐G, 1/2 in. needles
  • 1‐ml heparinized syringes with 23‐G, 1‐in. needles
  • 500‐µl EDTA‐coated microtainer tubes
  • Microtiter plate reader
  • Semi‐logarithmic graph paper
  • Additional reagents and equipment for injection of the mouse (Donovan and Brown, ), euthanasia of the mouse (Donovan and Brown, ), and blood collection via cardiac puncture (Donovan and Brown, )

Basic Protocol 3: Measurement of Regulated Exocytosis Using the Passive Systemic Anaphylaxis (PSA) Method

  • 250 to 500 µg/ml IgE anti‐DNP antibody (Sigma‐Aldrich) or IgE anti‐TNP (BD Pharmingen) in sterile PBS ( appendix 2A)
  • Antigen in 2% (w/v) Evans Blue (see recipe)
  • Formamide
  • Rectal temperature probe connected to a digital recorder (Cole‐Parmer)
  • 70‐µm nylon cell strainer (BD Falcon)
  • Spectrophotometer

Alternate Protocol 3: Detection of Regulated Exocytosis by Passive Systemic Anaphylaxis

  • 1.5 µg/ml Bordetella pertussis toxin (Invitrogen) in PBS ( appendix 2A)
  • 2.5 mg/ml DNP‐KLH and 0.5% (w/v) aluminum hydroxide (Rehydragel, Reheis Inc.; http://www.reheis.com) in PBS ( appendix 2A)
  • 0.5% (w/v) aluminum hydroxide (Rehydragel, Reheis Inc.; http://www.reheis.com) in PBS ( appendix 2A)
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Figures

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Literature Cited

Literature Cited
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