Measurement of Natural Killer Cell Progenitor Activity in Culture

Taku Kouro1, Takafumi Yokota1, Robert Welner1, Paul W. Kincade1

1 Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.3
DOI:  10.1002/0471142735.im22f03s66
Online Posting Date:  May, 2005
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Abstract

Natural killer cells are morphologically related to and have progenitors in common with B and T lymphocytes. In this unit, protocols describe how to induce natural killer cell differentiation from prolymphocytes. Since NK cell differentiation cultures also support B cell differentiation, this protocol is extremely useful for seeking the B and NK cell branch point in their differentiation pathway.

Keywords: Natural killer cells; progenitors; differentiation; culture; cytokine

     
 
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Table of Contents

  • Basic Protocol 1: Serum‐Free, Stromal‐Cell‐Free Natural Killer Cell Differentiation Culture
  • Alternate Protocol 1: Two‐Step Culture for Natural Killer Cell Differentiation
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Serum‐Free, Stromal‐Cell‐Free Natural Killer Cell Differentiation Culture

  Materials
  • Recombinant mouse IL‐15 (Research Diagnostics)
  • Staining/wash buffer (unit 22.2)
  • PE‐conjugated anti–pan NK cell marker CD49b(DX5) MAb (BD Pharmingen) or PE‐conjugated NK‐1.1 MAb (BD Pharmingen)
  • FITC‐conjugated anti‐CD3 MAb (2C11; BD Pharmingen)
  • Biotinylated anti‐CD19 MAb (BD Pharmingen)
  • APC‐conjugated anti‐CD11b(Mac‐1) MAb (BD Pharmingen)
  • Streptavidin‐PE‐Texas Red (Caltag)
  • Additional materials and equipment for preparing stromal‐cell‐free B cell differentiation cultures (unit 22.2, Basic Protocol 1), titration of cytokines (unit 22.1, Support Protocol), cell counting ( appendix 3B), and flow cytometry (Chapter 5)

Alternate Protocol 1: Two‐Step Culture for Natural Killer Cell Differentiation

  • Complete X‐VIVO15 serum‐free culture medium containing appropriate concentrations of stem cell factor, flt3‐ligand, and IL‐7 (unit 22.2)
  • Lin c‐kitlow prolymphocytes (unit 22.1)
  • Sterile wash buffer: combine 500 ml PBS ( appendix 2A) with 15 ml fetal bovine serum (FBS); filter through 0.22‐µm filter (store up to 1 year at 4°C)
  • PE‐conjugated anti‐CD122/IL‐2Rβ MAb (BD Pharmingen)
  • Complete X‐VIVO15 serum‐free culture medium containing appropriate concentration of IL‐15 (unit 22.2)
  • 6‐well tissue and 24‐well culture plates
  • Fluorescence activated cell sorter (e.g., MoFlo from Cytomation)
  • Additional reagents and equipment for B cell differentiation culture (unit 22.2)
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Figures

Videos

Literature Cited

Literature Cited
   Ikawa, T., Kawamoto, H., Fujimoto, S., and Katsura, Y. 1999. Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay. J. Exp. Med. 190:1617–1626.
   Kouro, T., Kumar, V., and Kincade, P.W. 2002. Relationships between early B‐ and NK‐lineage lymphocyte precursors in bone marrow. Blood 100:3672–3680.
   Kubota, A., Kubota, S., Lohwasser, S., Mager, D.L., and Takei, F. 1999. Diversity of NK cell receptor repertoire in adult and neonatal mice. J. Immunol. 163:212–216.
   Williams, N.S., Moore, T.A., Schatzle, J.D., Puzanov, I.J., Sivakumar, P.V., Zlotnik, A., Bennett, M., and Kumar, V. 1997. Generation of lytic natural killer 1.1+, Ly‐49– cells from multipotential murine bone marrow progenitors in a stroma‐free culture: Definition of cytokine requirements and developmental intermediates. J. Exp. Med. 186:1609‐1614.
   Williams, N.S., Klem, J., Puzanov, I.J., Sivakumar, P.V., Bennett, M., and Kumar, V. 1999. Differentiation of NK1.1+, Ly49+ NK cells from flt3+ multipotent marrow progenitor cells. J. Immunol. 163:2648‐2656.
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