Isolation of Mouse Intrahepatic Lymphocytes

I. Nicholas Crispe1

1 Yale University Medical School, New Haven, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.21
DOI:  10.1002/0471142735.im0321s22
Online Posting Date:  May, 2001
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The liver is a distinctive immunological environment in which are found a wide variety of cell types. The absolute number of intrahepatic lymphocytes (IHL) and the frequency of the various components of the cell mixture in the liver are influenced by the age and strain of the mouse, as well as by the presence of hormones, cytokines, and pathogens. The bulk of the liver consists of hepatocytes. In addition to IHL, the liver also contains a macrophage population, the Kupffer cells, and sinusoidal endothelial cells. The protocols described here can be used to deplete the liver tissue of hepatocytes, endothelial cells, and red blood cells, leaving a cell suspension consisting mainly of IHL in which the major contaminant is Kupffer cells (which can be removed by adherence to plastic). This unit provides two protocols that may be used to isolate IHL. One can be used to isolate IHL from multiple livers in parallel, whereas the more elaborate alternate protocol yields more cells per liver but is more appropriately used to recover the IHL from a single liver.

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Table of Contents

  • Basic Protocol 1: Basic Method for Isolation of IHL
  • Alternate Protocol 1: High‐Yield Method for Isolation of IHL
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Basic Method for Isolation of IHL

  • Mouse
  • PBS ( appendix 2A), ice cold
  • Tissue culture medium (e.g., RPMI 1640) with and without 5% FBS, ice cold
  • recipeDigestion medium (see recipe), freshly made on the day of experiment
  • 40% (w/v) metrizamide (Calbiochem or Boehringer Mannheim) in PBS
  • Tissue culture medium (e.g., RPMI 1640) with 10% FBS (optional)
  • 5‐ml syringe with 25‐G needle
  • Sieve (e.g., stainless steel gauze or metal tea‐strainer)
  • Plunger (e.g., ground‐glass plunger from a tissue homogenizer or a 10‐ml syringe plunger)
  • Refrigerated centrifuge (e.g., IEC Centra 8R with standard rotor)
  • Reciprocating water bath (e.g., Bio‐Rad Hot Shaker)
  • 10‐cm petri dishes (for tissue culture; optional)
  • Additional reagents and equipment for euthanasia using CO 2 (unit 1.8)

Alternate Protocol 1: High‐Yield Method for Isolation of IHL

  • recipeHanks' solutions A and B (see recipe)
  • Heparin
  • 37°C water bath
  • 95% O 2/5% CO 2 cylinder
  • Variable peristaltic pump (e.g., Myra Varipump)
  • Temperature‐controlled water recirculator (e.g., Haake FE2)
  • Knotes solvent‐recovery condenser (Fisher)
  • Portex tubing (to connect elements of the perfusion setup)
  • 4/0 surgical silk
  • Pediatric IV cannula, 22 to 24 G
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Literature Cited

Literature Cited
   Crispe, I.N. and Mehal, W.Z. 1996. Strange brew: T cells in the liver. Immunol. Today. 17:522‐525.
   Huang, L., Sye, K., and Crispe, I.N. 1994a. Proliferation and apoptosis of abundant B220+ CD4− CD8− T cells in the normal mouse liver. Int. Immunol. 6:533‐540.
   Huang, L., Soldevila, G., Leeker, M., Flavell, R.A., and Crispe, I.N. 1994b. Liver is the site of T cell destruction during peripheral deletion. Immunity. 1:741‐749.
   Itoh, H., Abo, T., Sugawara, S., Kanno, A., and Kumagai, K. 1988. Age related variation in the proportion and activity of murine liver natural killer cells and their cytotoxicity against regenerating hepatocytes. J. Immunol. 141:315‐327.
   MacDonald, H.R. 1995. NK1.1+ T cell receptor αβ cells: New clues to their origin, specificity and function. J. Exp. Med. 182:633‐638.
   Masuda, T., Ohteki, T., Abo, T., Seki, S., Nose, S., Nagura, H., and Kumagai, K. 1991. Expansion of the population of double negative CD4‐8, ‐ T αβ cells in the liver is a common feature of autoimmune mice. J. Immunol. 147:2907‐2912.
   Ohteki, T., Okuyama, R., Seki, S., Abo, T., Sugiura, K., Kusumi, A., Ohmori, T., Watanabe, H., and Kumagai, K. 1992. Age‐dependent increase of extrathymic T cells in the liver and their appearance in the periphery of older mice. J. Immunol. 149:1562‐1570.
   Sato, K., Ohtsuka, K., Hasegawa, K., Yamagiwa, S., Watanabe, H., Asakura, H., and Abo, T. 1995. Evidence for extrathymic generation of intermediate T cell receptor cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation. J. Exp. Med. 182:759‐767.
   Seki, S., Abo, T., Masuda, T., Ohteki, T., Kanno, A., Takeda, K., Rikiishi, H., Nagura, H., and Kumagai, K. 1990. Identification of activated T cell receptor γδ lymphocytes in the liver of tumor‐bearing hosts. J. Clin. Invest. 86:409‐415.
   Watanabe, H., Myaji, C., Kawachi, Y., Iiai, T., Ohtsuka, K., Iwanagi, T., Takahashi‐Iwanaga, H., and Abo, T. 1995. Relationships between intermediate TCR cells and NK1.1+ T cells in various immune organs. J. Immunol. 155:2972‐2983.
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