Isolation of Tissue Mast Cells

Marianna Kulka1, Dean D. Metcalfe2

1 National Research Council Canada, Institute for Nutrisciences and Health, Charlottetown, Prince Edward, Island, Canada, 2 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 7.25
DOI:  10.1002/0471142735.im0725s90
Online Posting Date:  August, 2010
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Abstract

Located primarily in tissues, mast cells are one of the principal effector cells in allergic inflammation. Mast cells derive from mononuclear precursor cells which undergo their final phase of differentiation in the tissues. Mast cells express a unique set of proteases and display functional diversity depending on the tissue in which they differentiate—a phenomenon often referred to as mast cell heterogeneity. Enzymatic digestion and density centrifugation have often been used to isolate human mast cells from tissues such as lung and skin, frequently resulting in cells with low viability and purity. Here, we describe a protocol that combines gentle enzymatic digestion with positive selection techniques to isolate reasonably viable and substantially enriched preparations of tissue mast cells. Curr. Protoc. Immunol. 90:7.25.1‐7.25.11. © 2010 by John Wiley & Sons, Inc.

Keywords: mast cells; human mast cells; lung, skin; fluorescent activated cell sorting (FACS); magnetic bead columns

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Skin Mast Cells
  • Alternate Protocol 1: Fluorescence‐Activated Cell Sorting (FACS) of Skin Mast Cells Using Expression of FcɛRI and Kit
  • Alternate Protocol 2: Enrichment of Skin Mast Cells by Positive Selection Immunomagnetic Separation
  • Basic Protocol 2: Isolation of Human Lung Mast Cells
  • Support Protocol 1: Acid Toluidine Blue Staining of Mast Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Isolation of Skin Mast Cells

  Materials
  • Skin samples: fresh skin segments stored in sterile PBS (Invitrogen) at 4°C or on ice
  • Serum‐free RPMI medium (see recipe for complete RPMI medium, but omit FBS)
  • Protease solution (see recipe)
  • PBS/DNase solution: 0.015 mg/ml bovine DNase I (Sigma‐Aldrich, cat. no. DN25) in sterile PBS (Invitrogen)
  • Complete RPMI medium (see recipe)
  • Lysis buffer (see recipe)
  • RPMI/DNase solution: 0.015 mg/ml DNase I (Sigma‐Aldrich, cat. no. DN25) in sterile serum‐free RPMI (see recipe for complete RPMI medium, but omit FBS)
  • 70% isotonic Percoll (with 30% RPMI/DNase; see recipe)
  • Recombinant human stem cell factor (Peprotech)
  • 100× 15–mm petri dishes (Falcon)
  • 50‐ and 15‐ml conical graduated polypropylene centrifuge tubes (Falcon)
  • Tweezers, scissors, and scalpels, kept in sterile beaker with 70% ethanol
  • Tabletop centrifuge
  • Platform shaker
  • 70‐µm cell strainer (Falcon)
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A), counting viable cells by trypan blue exclusion ( appendix 3B), and determining mast cell purity by toluidine blue staining ( protocol 5)

Alternate Protocol 1: Fluorescence‐Activated Cell Sorting (FACS) of Skin Mast Cells Using Expression of FcɛRI and Kit

  • Phosphate‐buffered saline (PBS; Invitrogen) containing 0.1% (w/v) bovine serum albumin (BSA), 4°C
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Anti‐FcɛRI‐APC (eBioscience)
  • Mouse IgG‐APC (eBioscience)
  • Anti‐CD117‐PE (BD Pharmingen)
  • Mouse IgG 1‐PE (BD Pharmingen)
  • 5‐ml polystyrene round‐bottom tubes
  • Refrigerated centrifuge
  • Additional reagents and equipment for flow cytometry (Chapter 5)

Alternate Protocol 2: Enrichment of Skin Mast Cells by Positive Selection Immunomagnetic Separation

  • Phosphate‐buffered saline (PBS; Invitrogen) containing 0.1% (w/v) bovine serum albumin (BSA), 4°C
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • Anti‐PE MicroBeads and wash buffer (Miltenyi Biotec)
  • Anti‐FcɛRI‐ APC (eBioscience)
  • Mouse IgG‐APC (eBioscience)
  • Anti‐CD117‐PE (BD Pharmingen)
  • Mouse IgG 1‐PE (BD Pharmingen)
  • MACS column and magnet (Miltenyi Biotec)
  • Additional reagents and equipment for trypan blue exclusion test for cell viability ( appendix 3B) and toluidine blue staining for mast cell purity ( protocol 5)

Basic Protocol 2: Isolation of Human Lung Mast Cells

  Materials
  • Fresh lung tissue stored in sterile PBS at 4°C or on ice
  • Modified Tyrode's buffer (see recipe)
  • 0.5 mg/ml collagenase in sterile PBS
  • PBS/DNase solution: 0.015 mg/ml bovine DNase I (Sigma‐Aldrich) in sterile PBS (Invitrogen)
  • Lysis buffer (see recipe)
  • RPMI/DNase solution: 0.015 mg/ml bovine DNase I (Sigma‐Aldrich) in serum‐free RPMI medium (see recipe for complete RPMI medium, but omit FBS)
  • 70% isotonic Percoll (with 30% RPMI/DNase see recipe)
  • Complete RPMI medium (see recipe)
  • Recombinant human stem cell factor (Peprotech)
  • 100 × 15–mm petri dishes (Falcon)
  • Tweezers, scissors, and scalpels, kept in sterile beaker with 70% ethanol
  • 50‐ and 15‐ml conical graduated polypropylene centrifuge tubes (Falcon)
  • Platform shaker
  • 70‐µm cell strainer
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A), counting viable cells by trypan blue exclusion ( appendix 3B), and determining mast cell purity by toluidine blue staining ( protocol 5)

Support Protocol 1: Acid Toluidine Blue Staining of Mast Cells

  Materials
  • Mota's fixative (see recipe)
  • 66% and 100% ethanol
  • Acid toluidine blue solution (see recipe)
  • Cytocentrifuge (Cytospin, Shandon/Lipshaw)
  • Microscope slides
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Figures

Videos

Literature Cited

Literature Cited
   Bischoff, S.C. 2009. Physiological and pathophysiological functions of intestinal mast cells. Semin. Immunopathol. 31:185‐205.
   Church, M.K. and Clough, G.F. 1999. Human skin mast cells: In vitro and in vivo studies. Ann. Allergy Asthma Immunol 83:471‐475.
   Dvorak, A.M., Mitsui, H., and Ishizaka, T. 1993. Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c‐kit ligand; distinction from mature basophilic leukocytes undergoing secretion in the same cultures. J. Leukoc. Biol. 54:465‐485.
   Kirshenbaum, A.S. 2000. Regulation of mast cell number and function. Hematol. Oncol. Clin. North Am. 14:497‐516.
   Kirshenbaum, A.S., Goff, J.P., Semere, T., Foster, B., Scott, L.M., and Metcalfe, D.D. 1999. Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c‐kit(+), and expresses aminopeptidase N (CD13). Blood 94:2333‐2342.
   Mekori, Y.A. and Metcalfe, D.D. 1999. Mast cell–T cell interactions. J. Allergy Clin. Immunol. 104:517‐523.
   Mikhail, G.R. and Miller‐Milinska, A. 1964. Mast cell population in human skin. J. Invest. Dermatol. 43:249‐254.
   Miller, G.R. and Pemberton, A.D. 2002. Tissue‐specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut. Immunology 105:375‐390.
   Nilsson, G., Blom, T., Kusche‐Gullberg, M., Kjellen, L., Butterfield, J.H., Sundstrom, C., Nilsson, K., and Hellman, L. 1994. Phenotypic characterization of the human mast cell line HMC‐1. Scand. J. Immunol. 39:489‐498.
   Okayama, Y., Benyon, R.C., Rees, P.H., Lowman, M.A., Hillier, K., and Church, M.K. 1992. Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine. Clin. Exp. Allergy 22:401‐409.
   Reid, A.C., Silver, R.B., and Levi, R. 2007. Renin: At the heart of the mast cell. Immunol. Rev. 217:123‐140.
   Sellge, G. and Bischoff, S.C. 2006. Isolation, culture, and characterization of intestinal mast cells. Methods Mol. Biol. 315:123‐138.
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