Isolation of Mononuclear Cells from Tonsillar Tissue

Andrew Johnston1, Sigrun L. Sigurdardottir2, Judith J. Ryon3

1 Department of Dermatology, University of Michigan, Ann Arbor, Michigan, 2 Department of Immunology, Landspitali‐University Hospital, Reykjavik, Iceland, 3 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.8
DOI:  10.1002/0471142735.im0708s86
Online Posting Date:  August, 2009
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Located on the inside of the throat, the paired palatine tonsils form part of the first major barrier protecting the digestive and respiratory tracts from potentially invading microorganisms. The tonsils have a surface of stratified squamous epithelium that extends into deep and branched crypts lined by reticulated epithelium, which in parts may only be one cell thick. Organized in the sub‐epithelial space are B cell rich lymphoid follicles. T cells are mostly located in the extra‐follicular spaces with a very high CD4:CD8 T cell ratio. In addition to the T and B cell subsets, six phenotypes of dendritic cells (DC) have been identified in the tonsils: Langerhans cells in the squamous epithelium, germinal center DC, and follicular DC in the germinal center, and another three DC phenotypes that are located in the extra‐follicular area (interdigitating DC, plasmacytoid DC, and lympho‐epithelial symbiosis‐DC). Here, we describe the isolation of tonsil mononuclear cells from fresh human tonsil. Curr. Protoc. Immunol. 86:7.8.1‐7.8.4. © 2009 by John Wiley & Sons, Inc.

Keywords: tonsils; T cells; B cells; lymphocytes

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1:

  • Fresh human palatine tonsils
  • Isotonic saline or Hanks balanced salt solution (HBSS) with antibiotics (see recipe), 4°C
  • Complete RPMI medium supplemented with 10% (v/v) human AB serum ( appendix 2A) with 5 µg/ml gentamicin and 0.5 µg/ml amphotericin B
  • Forceps, sterile
  • 150 × 25–mm petri dishes
  • Disposable sterile scalpels (Swann‐Morton, cat. no. 0511 or equivalent)
  • Straight iris scissors (Fisher, cat. no. S17338 or equivalent)
  • Dissecting forceps (VWR, cat. no. 382027‐384 or equivalent)
  • 3‐in. stainless steel sieve with 250‐µm mesh (Thomas Scientific, cat. no. 8323‐N48 or equivalent)
  • 5‐ml plastic syringe plunger
  • 50‐ml polycarbonate centrifuge tubes
  • 3‐ml transfer pipet (single‐use Pasteur pipets; Sarstedt, cat. no. 861171)
  • 40‐µm plastic cell strainer (Fisher, cat. no. 22363547)
  • Low‐speed centrifuge with fixed‐angle or swinging‐bucket rotor (e.g., Sorvall RT‐6000B with H‐1000B rotor)
  • Additional reagents and equipment for Ficoll‐Hypaque cell separation (unit 7.1) and cryopreservation of cells ( appendix 3G)
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Literature Cited

   Hart, D. and McKenzie, J. 1988. Isolation and characterization of human tonsil dendritic cells. J. Exp. Med. 168:157‐170.
   Hoffmann, M., Schmidt, D., and Oettgen, H. 1973. Production of antibody to sheep red blood cells by human tonsil cells in vitro. Nature 243:408‐410.
   Muraguchi, A., Butler, J., Kehrl, J., and Fauci, A.S. 1983. Differential sensitivity of human B cell subsets to activation signals delivered by anti‐µ antibody and proliferative signals delivered by a monoclonal B cell growth factor. J. Exp. Med. 157:530‐546.
   Nakagawa, T., Nakagawa, N., Ambrus, J.L., and Fauci, A.S. 1988. Differential effects of Interleukin 2 vs. B cell growth factor on human B cells. J. Immunol. 140:465‐469.
   Sloyer, J., Veltri, R., and Sprinkle, P. 1973. In vitro IgM antibody synthesis by human tonsil‐derived lymphocytes. J. Immunol. 111:183‐188.
   Summers, K.L., Hock, B.D., McKenzie, J.L. and Hart, D.N. 2001. Phenotypic characterization of five dendritic cell subsets in human tonsils. Am. J. Pathol. 159:285‐295.
   Strowig, T., Brilot, F., Array, F., Bougras, G., Thomas, D., Muller, W.A., and Munz, C. 2008. Tonsillar NK cells restrict B cell transformation by the Epstein‐Barr virus via IFN‐gamma. PloS Pathog. 4:e27.
   Watanabe, T., Yoshizaki, K., Yagura, T., and Yamamura, Y. 1974. In vitro antibody formation by human tonsil lymphocytes. J. Immunol. 113:608‐616.
Key Reference
   Watanabe et al., 1974. See above.
  Describes protocol and expected yields, as well as the determination of optimal conditions for in vitro synthesis of immunoglobulin by tonsil lymphocytes.
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