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Isolation of Murine Natural Killer Cells

Amy Reichlin¹, Koho Iizuka¹, Wayne M. Yokoyama¹

¹Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 3.22

DOI:

10.1002/0471142735.im0101s35

Online Posting Date:

May, 2001

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Abstract

This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by cytotoxic depletion of contaminating cells with selected monoclonal antibodies (MAbs), complement lysis, and density-gradient centrifugation to eliminate dead cells. The advantage of this negative selection process is that the NK cells are not coated with antibody and, therefore, are not at risk of activation by antibody cross-linking. Purity can then be assessed by cell surface phenotype.

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Unit Introduction
Basic Protocol: Preparation of NK Cells from Mouse Spleen
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Preparation of NK Cells from Mouse Spleen

Materials

C57BL/6- or C57BL/10 mice
Tris/NH₄Cl lysing buffer (see recipe)
HBSS/3% FBS (see recipe)
10× stocks of MAbs GK1.5 (ATCC TIB 207), 53-6.72 (TIB 105), M5/114.15.2 (TIB 120), and J11d.2 (TIB 183; PharMingen; see recipe for MAb solutions)
MAb MAR18.5 (TIB 216; PharMingen) at 1 mg/ml (see recipe for MAb solutions)
HBSS with phenol red (Life Technologies)/10% FBS (see recipe)
Sterile reconstituted Low-Tox-M rabbit complement (see recipe)
Lympholyte-M density gradient (Accurate Chemical and Scientific), room temperature
Complete RPMI-10 medium (appendix 2A)
Anti-CD3-FITC and PK136-PE conjugates (PharMingen)

50- and 15-ml conical screw-cap polypropylene centrifuge tubes
Beckman CS-6R swinging-bucket centrifuge (or equivalent)
Serologic pipets

Additional reagents and equipment for preparation of a cell suspension from mouse spleen (unit 3.1), red blood cell lysis (unit 3.1), counting of viable cells by trypan blue exclusion (appendix 3B), and FACS analysis (Chapter 5.0)

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Figures

Figure 3.22.1

Two-color FACS analysis of purified NK cells. From unfractionated C57BL/6-responding spleen cells composed of 2.5% NK1.1⁺ CD3^–, the protocol routinely results in a population that is ~80% to 90% NK1.1⁺ CD3^– and ~5% to 10%CD3⁺ T cells. A phycoerythrin-conjugated anti-NK1.1 antibody (clone PK136, PharMingen) and a FITC-anti-CD3 antibody (clone 145-2C11) were used to stain cells for analysis with a FACSCalibur (Becton Dickinson).

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Figure 3.22.2

Killing profile of purified NK cells compared to unfractionated splenocytes against labeled YAC-1 targets in 4 hr ⁵¹Cr-release assay.

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Literature Cited

Literature Cited
	Bancroft, G. J. 1993. The role of natural killer cells in innate resistance to infection. Curr. Opin. Immunol. 5:503-510.
	Bendelac, A. 1995. Mouse NK1⁺ T cells. Curr. Opin. Immunol. 7:367-374.
	Denkers, E. Y., Gazzinelli, R. T., Martin, D., and Sher, A. 1993. Emergence of NK1.1⁺ cells as effectors of IFN- dependent immunity to Toxoplasma gondii in MHC class I–deficient mice. J. Exp. Med. 178:1465-1472.
	Hackett, J. Jr., Tutt, M., Lipscomb, M., Bennett, M., Koo, G., and Kumar, V. 1986. Origin and differentiation of natural killer cells. II. Functional and morphologic studies of purified NK-1.1⁺ cells. J. Immunol. 136:3124-3131.
	Herberman, R. 1982. NK Cells and Other Natural Effector Cells. Academic Press, New York.
	Karlhofer, F. M. and Yokoyama, W. M. 1991. Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways. J. Immunol. 14:3662-3673.
	Morelli, L., Lusignan, Y., and Lemieux, S. 1992. Heterogeneity of natural killer cell subsets in NK-1.1⁺ and NK-1.1–inbred mouse strains and their progeny. Cell. Immunol. 141:148-160.
	Patel, M. R. and Linna, T. J. 1984. Enrichment of mouse splenic natural killer cells using discontinuous polyvinylpyrrolidone silca (Percoll) gradients. Immunology. 53:721-729.
	Ravnik, S. E., Gage, S., and Pollack, S. B. 1988. Self-generating density gradients of Percoll provide a simple and rapid method that consistently enriches natural killer cells. J. Immunol. Methods. 110:161-168.
	Seaman, W. E., Sleisenger, M., Eriksson, E., and Koo, G. C. 1987. Depletion of natural killer cells in mice by monoclonal antibody to NK-1.1. Reduction in host defense against malignancy without loss of cellular or humoral immunity. J. Immunol. 138:4539-4544.
	Timonen, T., Ortaldo, J. R., and Herberman, R. B. 1981. Characteristics of human large granular lymphocytes and relationship to natural killer and K cells. J. Exp. Med. 153:569-582.
	Trinchieri, G. 1989. Biology of natural killer cells. Adv. Immunol. 47:187-376.
	Tripp, C. S., Wolf, S. F., and Unanue, E. R. 1993. Interleukin 12 and tumor necrosis factor are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. U. S.A. 90:3725-3729.