Isolation of Murine Natural Killer Cells

Melissa A. Pak‐Wittel1, Sytse J. Piersma1, Beatrice F. Plougastel1, Jennifer Poursine‐Laurent1, Wayne M. Yokoyama2

1 Division of Rheumatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, 2 Howard 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.im0322s105
Online Posting Date:  April, 2014
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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 depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross‐linking. Additionally, negative selection provides a way to isolate diverse subpopulations of NK cells without selectively purifying a specific subpopulation. Following enrichment, NK cell purity can be assessed by cell surface phenotype using flow cytometry. Curr. Protoc. Immunol.. 105:3.22.1‐3.22.9. © 2014 by John Wiley & Sons, Inc.

Keywords: immunology; innate immunity; cell isolation; magnetic separation

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Table of Contents

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

  • C57BL/6‐ or C57BL/10 mice
  • Tris/NH 4Cl lysing buffer (see recipe)
  • Complete RPMI‐10 (R10) medium (see recipe)
  • Fc blocking buffer (see recipe) or a commercially available Fc‐blocking agent
  • Isolation buffer (see recipe)
  • 0.5 mg/ml biotinylated MAbs (eBioscience): CD3ε (145‐2C11), CD8a (53‐6.7), CD4 (GK1.5), CD14 (Sa2‐8), CD19 (eBio1D3), TER‐119, and Ly6G (GR‐1)
  • Anti‐biotin microbeads (Miltenyi Biotech)
  • Anti‐biotin Dynabeads for alternative separation protocol
  • Fluorophore‐conjugated MAbs for flow cytometry: CD3ε‐FITC, NK1.1‐(PK136)‐PerCP‐Cy5.5, CD19‐PE‐Cy7, MHC Class II (I‐A/I‐E)‐AlexaFluor700, CD11c‐APC‐Cy7, CD11b‐eFluor450 (eBioscience), Ly6G (1A8)‐PE and F4/80 (BM8)‐AlexaFluor647 (Biolegend)
  • Fixable cell viability marker eFluor506 for flow cytometry (eBiosciences)
  • 70‐µm cell strainer (BD Biosciences)
  • 50‐ and 15‐ml conical screw‐cap polypropylene centrifuge tubes
  • Beckman GS‐6R swinging‐bucket centrifuge with GH3.8 rotor (or equivalent)
  • Platform rocker
  • LS column and magnetic separator (Miltenyi Biotec; also see unit 3.5)
  • DynaMag‐15 (Life Technologies) for alternate separation protocol.
  • 40‐µm pre‐separation filter (Miltenyi Biotec)
  • Additional reagents and equipment for euthanasia of mice (unit 1.8), removal of mouse spleen (unit 1.9), preparation of a cell suspension from mouse spleen (unit 3.1), red blood cell lysis (unit 3.1), use of nylon wool column for removing macrophages and dendritic cells (unit 3.2), counting of viable cells by trypan blue exclusion ( appendix 3B), antibody titration (unit 5.3), magnetic bead separation (unit 3.5), and flow cytometry (Chapter 5)
NOTE: All reagents and materials used in the preparation of these cells must be sterile if cells are to be used in culture.
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Literature Cited

Literature Cited
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  Hackett, J., 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.
  Horowitz, A., Stegmann, K.A., and Riley, E.M. 2011. Activation of natural killer cells during microbial infections. Front. Immunol. 2:88.
  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. 146:3662‐3673.
  Kim, S., Iizuka, K., Kang, H.‐S.P., Dokun, A., French, A.R., Greco, S., and Yokoyama, W.M. 2002. In vivo developmental stages in murine natural killer cell maturation. Nat. Immunol. 3:523‐528.
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  Meinhardt, K., Kroeger, I., Abendroth, A., Müller, S., Mackensen, A., and Ullrich, E. 2012. Influence of NK cell magnetic bead isolation methods on phenotype and function of murine NK cells. J. Immunol. Methods 378:1‐10.
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  Patel, M.R. and Linna, T.J. 1984. Enrichment of mouse splenic natural killer cells using discontinuous polyvinylpyrrolidone silica (Percoll) gradients. Immunology 53:721‐729.
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  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.
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