Immunophenotypic Analysis of Human Mast Cells by Flow Cytometry

Luis Escribano1, Raquel Navalón1, Rosa Núñez1, Beatriz Díaz Agustín1, Pilar Bravo1

1 Hospital Ramón y Cajal, Mast Cell Unit, Madrid, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.6
DOI:  10.1002/0471142956.cy0606s12
Online Posting Date:  May, 2001
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Abstract

The immunophenotypic identification and enumeration of human bone marrow mast cells represents a clear demonstration of the utility of flow cytometry for rare‐event analysis. The basic approach can be applied to a variety of specimens, including bone marrow, peripheral blood, ascitic fluid, and lymphoid tissue such as adenoids. Special emphasis is placed on markers with potential utility for distinguishing between normal, reactive, and pathological mast cells. From the clinical aspect, immunophenotypic analysis of mast cells may have great utility in supporting the diagnosis of tissue involvement in mastocytosis.

     
 
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Table of Contents

  • Basic Protocol 1: Identification and Enumeration of Bone Marrow Mast Cells (BMMCs)
  • Basic Protocol 2: Identification and Enumeration of Ascitic Fluid Mast Cells
  • Basic Protocol 3: Identification and Enumeration of Lymphoid Tissue Mast Cells
  • Support Protocol 1: Immunophenotypical Characterization of Mast Cells
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Identification and Enumeration of Bone Marrow Mast Cells (BMMCs)

  Materials
  • Source of BM samples
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.5% (w/v) toluidine blue in methanol, pH 3.0
  • Phycoerythrin (PE)‐conjugated CD117 monoclonal antibody (CD117‐PE MAb), appropriately titered
  • Fluorescein isothiocyanate (FITC)‐conjugated anti‐IgE monoclonal or polyclonal Ab (anti‐IgE‐FITC Ab) or FITC‐conjugated CD33 MAb(CD33‐FITC), appropriately titered
  • FITC‐conjugated CD34 MAb (CD34‐FITC), appropriately titered
  • PE‐cyanine 5 (PE‐Cy5 tandem fluorochrome)–conjugated CD45 (CD45‐PE‐Cy5 MAb) or peridin chlorophyll protein (PerCP)–conjugated CD45 MAb (CD45‐PerCP MAb), appropriately titered
  • 1× ammonium chloride lysing solution ( appendix 2A), freshly prepared, or 1× Quicklysis (IMICO)
  • PBS containing 1% paraformaldehyde (optional)
  • 14‐ to 8‐G biopsy needle
  • Vacutainer tubes containing either tripotassium EDTA or heparin
  • 25‐G needle and syringe
  • Hematology analyzer
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer with at least three fluorescence detectors and appropriate filter sets for detection of FITC, PE, and PE‐Cy5 or PerCP
  • Software for analysis of flow cytometry data: e.g., Paint‐A‐Gate Pro (Becton Dickinson)
NOTE: Each vial of anti‐IgE Ab should be stored frozen in aliquots. Each aliquot should be titrated before use (basophils should be used as target positive control cells) and kept at 4° to 8°C until finished.

Basic Protocol 2: Identification and Enumeration of Ascitic Fluid Mast Cells

  Materials
  • Ascitic fluid
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 50‐ml centrifuge tubes (e.g., Falcon, Becton Dickinson)
  • Hematology analyzer
  • Additional reagents and equipment for identification and enumeration of BMMCs (see protocol 1)

Basic Protocol 3: Identification and Enumeration of Lymphoid Tissue Mast Cells

  Materials
  • Fresh samples from lymphoid tissue (tonsils, adenoids, lymph nodes)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 90‐mm petri dishes
  • Surgical blades
  • Tweezers
  • Mechanical disaggregation system (e.g., Medimachine with 50‐µl Medicons; Dako)
  • Additional reagents and equipment for identification and enumeration of BMMCs (see protocol 1)

Support Protocol 1: Immunophenotypical Characterization of Mast Cells

  Materials
  • Appropriately titered FITC‐conjugated monoclonal antibodies: CD2, CD25, CD35, CD69, and anti‐bcl2
  • Samples of bone marrow (see protocol 1, steps to ), ascites fluid (see protocol 2, steps to ), or lymphoid tissue (see protocol 3, steps to )
  • Reagents for cell fixation and permeabilization to assess the cytoplasmic expression of the bcl‐2 protein (e.g., Fix & Perm, Caltag; Intrastain, Dako; or Intraprep, Immunotech)
  • 12 × 75–mm polystyrene tubes
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Figures

Videos

Literature Cited

Literature Cited
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