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Isolation of Mouse Intrahepatic Lymphocytes

I. Nicholas Crispe¹

¹Yale 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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Abstract

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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Unit Introduction
Basic Protocol: Basic Method for Isolation of IHL
Alternate Protocol: High-Yield Method for Isolation of IHL
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol: Basic Method for Isolation of IHL

Materials

Mouse
PBS (appendix 2A), ice cold
Tissue culture medium (e.g., RPMI 1640) with and without 5% FBS, ice cold
Digestion 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 CO₂ (unit 1.8)

Alternate Protocol: High-Yield Method for Isolation of IHL

Additional Materials (also see Basic Protocol)

Hanks' solutions A and B (see recipe)
Heparin
37°C water bath
95% O₂/5% CO₂ 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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Figures

Figure 3.21.1

Detailed anatomy of the portal vein cannulation. The figure illustrates the placement of a flexible cannula, as described in the Alternate Protocol. The Basic Protocol uses a steel syringe needle, which need not be tied in.

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Figure 3.21.2

The perfusion apparatus used in the Alternate Protocol. The design allows warm, humidified 95% O₂/5% CO₂-equilibrated Hanks' A solution to be infused directly into the portal vein. Note the condenser, which rewarms the Hanks' solution immediately before it is infused.

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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 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 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 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.