Generation, Isolation, and Maintenance of Rodent Mast Cells and Mast Cell Lines

Bettina M. Jensen1, Emily J. Swindle1, Shoko Iwaki1, Alasdair M. Gilfillan1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.23
DOI:  10.1002/0471142735.im0323s74
Online Posting Date:  September, 2006
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Abstract

Antigen‐mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high‐affinity IgE receptor (FcɛRI) and, more recently, other receptors expressed on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone‐marrow‐derived mast cells, the rat basophilic leukemia cell line RBL‐2H3, and the mouse MC/9 mast cell line. In this unit, we describe the protocols used for the isolation and/or culture of these cells and discuss the relative merits of their use.

Keywords: FcɛRI; Mast Cells; Peritoneal mast cells; Mouse bone marrow‐derived mast cells (BMMC); RBL‐2H3 cells; MC/9 cells

     
 
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Table of Contents

  • Basic Protocol 1: Isolation and Culture of Peritoneal Mast Cells
  • Basic Protocol 2: Generation and Culturing of Mouse Bone Marrow‐Derived Mast Cells and Characterization by Flow Cytometry
  • Support Protocol 1: Splitting and Subculturing of RBL‐2H3 Mast Cell Line
  • Support Protocol 2: Splitting and Subculturing of MC/9 Mast Cell Line
  • Support Protocol 3: Startup of Cultures from Frozen Aliquots
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation and Culture of Peritoneal Mast Cells

  Materials
  • 10× and 1× Hanks' balanced salt solution (HBSS) without Ca2+ or Mg2+ (e.g., Invitrogen) with phenol red
  • Rat (150 to 200 g, 3 to 6 months old) or mouse (20 g, 2 to 6 months old)
  • 70% ethanol
  • 0.8% (w/v) M NH 4Cl in double‐distilled H 2O
  • 70% isotonic Percoll solution (see recipe)
  • PMC culture medium (see recipe)
  • 0.4% (w/v) trypan blue in PBS (see appendix 2A for PBS)
  • 0.05% (w/v) toluidine blue in PBS (see appendix 2A for PBS)
  • 10‐ml syringes (for mice) or 30‐ml syringes (for rats)
  • 23‐G needles (for mice) or 19‐G needles (for rats)
  • Dissecting instruments: scissors and forceps
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Centrifuge
  • 3‐ml pipet (Corning)
  • Additional equipment and reagents for euthanasia of rodents (unit 1.8), counting cells using a hemacytometer ( appendix 3A), trypan blue exclusion test of cell viability ( appendix 3B), and toluidine blue staining of mast cells (unit 7.25)
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.NOTE: All procedures are performed in a laminar flow hood. All reagents are sterilized by filtration and all surgical equipment sterilized by immersion in 70% ethanol prior to use.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Generation and Culturing of Mouse Bone Marrow‐Derived Mast Cells and Characterization by Flow Cytometry

  Materials
  • Mouse (2 to 6 months old)
  • 70% ethanol
  • BMMC–MC/9 culture medium (see recipe), prewarmed to 37°C
  • 0.05% (w/v) toluidine blue in PBS (see appendix 2A for PBS)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.1% (w/v) BSA, 4°C
  • Anti‐FcɛRI‐FITC (eBioscience, cat no. 11‐5898) and isotype control, hamster IgG‐FITC (eBioscience, cat. no. 11‐4444) or anti‐CD117‐PE (BD Pharmingen, cat. no. 555714) and isotype control, mouse IgG 1‐PE (BD Pharmingen, cat. no. 555749)
  • Dissecting instruments: scissors and forceps
  • 6‐well tissue culture plates (Costar)
  • 30‐ml syringes
  • 25‐G needles
  • 125‐cm2 tissue culture flask (Sarstsedt, Nunc)
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Flow cytometer tubes: 5‐ml polystyrene round‐bottom tubes
  • Flow cytometer (Chapter 5)
  • Additional reagents and equipment for euthanasia (unit 1.8), counting cells using a hemacytometer ( appendix 3A), toluidine blue staining of mast cells (unit 7.25), and flow cytometry (Chapter 5)
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.NOTE: All procedures, including bone removal and femur lavage, are performed in a laminar flow hood. All reagents are sterilized by filtration through an 0.22‐µm filter and all surgical equipment are sterilized by immersion in 70% ethanol prior to use.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Splitting and Subculturing of RBL‐2H3 Mast Cell Line

  Materials
  • Near‐confluent culture of RBL‐2H3 cells (ATCC no. CRL‐2256; see protocol 5 for instructions on defrosting cell sample and establishing culture)
  • RBL‐2H3 culture medium (see recipe)
  • 0.25% trypsin‐EDTA (e.g., Invitrogen)
  • RBL‐2H3 culture medium (see recipe) containing 10% (v/v) DMSO
  • Liquid nitrogen
  • Inverted microscope (optional)
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Tabletop centrifuge
  • Culture flasks
  • Cryotubes (e.g., Nunc)
  • Liquid nitrogen freezer (Dewar flask and canes to accommodate cryotubes)
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.

Support Protocol 2: Splitting and Subculturing of MC/9 Mast Cell Line

  Materials list
  • MC/9 cells (ATCC no. CRL‐8306; see protocol 5 for instructions on defrosting cell sample and establishing culture)
  • BMMC–MC/9 culture medium (see recipe)
  • BMMC–MC/9 culture medium (see recipe) containing 10% (v/v) DMSO
  • Liquid nitrogen
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Tabletop centrifuge
  • Culture flasks
  • Cryotubes (e.g., Nunc)
  • Liquid nitrogen freezer (Dewar flask and canes to accommodate cryotubes)
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.

Support Protocol 3: Startup of Cultures from Frozen Aliquots

  Materials
  • Frozen aliquot of cells ( protocol 3 or protocol 42)
  • Cell culture medium
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Tabletop centrifuge
  • Additional reagents and equipment for counting cells using a hemacytometer ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Ali, K., Bilancio, A., Thomas, M., Pearce, W., Gilfillan, A.M., Tkaczyk, C., Kuehn, N., Gray, A., Giddings, J., Peskett, E., Fox, R., Bruce, I., Walker, C., Sawyer, C., Okkenhaug, K., Finan, P., and Vanhaesebroeck, B. 2004. Essential role for the p110δ phosphoinositide 3‐kinase in the allergic response. Nature 431:1007‐1011.
   Barsumian, E.L., Isersky, C., Petrino, M.G., and Siraganian, R.P. 1981. IgE‐induced histamine release from rat basophilic leukemia cell lines: Isolation of releasing and nonreleasing clones. Eur. J. Immunol. 11:317‐323.
   Beaven, M.A. and Metzger, H. 1993. Signal transduction by Fc receptors: The FcɛRI case. Immunol. Today 14:222‐226.
   Coleman, J.W., Holliday, M.R., Kimber, I., Zsebo, K.M., and Galli, S.J. 1993. Regulation of mouse peritoneal mast cell secretory function by stem cell factor, IL‐3 or IL‐4. J. Immunol. 150:556‐562.
   Eastmond, N.C., Banks, E.M.S., and Coleman, J.W. 1997. Nitric oxide inhibits IgE‐mediated degranulation of mast cells and is the principal intermediate in IFN‐γ‐induced suppression of exocytosis. J. Immunol. 159:1444‐1450.
   Gilfillan, A.M., Kado‐Fong, H., Wiggan, G.A., Hakimi, J., Kent, U., and Kochan, J.P. 1992. Conservation of signal transduction mechanisms via the human FcɛRIα after transfection into a rat mast cell line, RBL 2H3. J. Immunol. 149:2445‐2451.
   Gilfillan, A.M. and Tkaczyk, C. 2006. Integrated signalling pathways for mast‐cell activation. Nat. Rev. Immunol. 6:218‐230.
   Ishizuka, T., Oshiba, A., Sakata, N., Terada, N., Johnson, G.L., and Gelfand, E.W. 1996. Aggregation of the FcɛRI on mast cells stimulates c‐Jun amino‐terminal kinase activity: A response inhibited by wortmannin. J. Biol. Chem. 271:12762‐12766.
   Jones, S.V., Choi, O.H., and Beaven, M.A. 1991. Carbachol induces secretion in a mast cell line (RBL‐2H3) transfected with the m1 muscarinic receptor gene. FEBS Lett. 289:47‐50.
   Kinet, J.P., Metzger, H., Hakimi, J., and Kochan, J. 1987. A cDNA presumptively coding for the α subunit of the receptor with high affinity for immunoglobulin E. Biochemistry 26:4605‐4610.
   Metcalfe, D.D., Baram, D., and Mekori, Y.A. 1997. Mast cells. Physiol. Rev. 77:1033‐1079.
   Nabel, G., Galli, S.J., Dvorak, A.M., Dvorak, H.F., and Cantor, H. 1981. Inducer T lymphocytes synthesize a factor that stimulates proliferation of cloned mast cells. Nature 291:332‐334.
   Repetto, B., Bandara, G., Kado‐Fong, H., Larigan, J.D., Wiggan, G.A., Pocius, D., Basu, M., Gilfillan, A.M., and Kochan, J.P. 1996. Functional contributions of the FcɛRIα and FcɛRIγ subunit domains in FcɛRI‐mediated signaling in mast cells. J. Immunol. 156:4876‐4883.
   Stevens, R.L., Friend, D.S., McNeil, H.P., Schiller, V., Ghildyal, N., and Austen, K.F. 1994. Strain‐specific and tissue‐specific expression of mouse mast cell secretory granule proteases. Proc. Natl. Acad. Sci. U.S.A. 91:128‐132.
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