Assays of Natural Killer (NK) Cell Ligation to Target Cells

Filippo Renò1, Stefano Papa1, Marco Vitale2, Loris Zamai3

1 University of Urbino, Urbino, Italy, 2 University of Brescia, Brescia, Italy, 3 University of Bologna, Bologna, Italy
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.10
DOI:  10.1002/0471142956.cy0910s04
Online Posting Date:  May, 2001
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Assays for natural killer cells have long been established in the flow repertoire, but successful application of a flow‐based protocol requires attention to many details. This unit provides these details in a comprehensive manner. In particular, the assay is designed for simple bench‐top cytometers, rather than for more complex research instruments. Following this protocol, even a novice user should be able to achieve successful completion of an NK assay.

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Table of Contents

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

  • Carbonyl iron (Sigma)
  • Heparinized human blood (unit 5.1)
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • Ficoll‐Hypaque (Seromed) or Percoll (Sigma; S = 1.077)
  • RPMI‐10 medium (see recipe)
  • K562 chronic myeloid leukemia cells grown in suspension in RPMI‐10 to logarithmic phase
  • Propidium iodide (PI) stock solution (see recipe)
  • Fluorochrome‐conjugated monoclonal antibodies (MAbs; e.g., PE‐conjugated anti‐CD16, FITC‐conjugated anti‐CD3, and PerCP‐conjugated anti‐CD8)
  • 37°C water bath
  • 15‐ and 50‐ml polypropylene centrifuge tubes (Falcon)
  • Beckman GS‐15R centrifuge (or equivalent)
  • Flow cytometer with 488‐nm light source (e.g., Becton Dickinson, Coulter, or Ortho)
  • Software for multiparametric analysis
  • Additional reagents and equipment for removing monocytes from blood (unit 5.1), cell culture ( appendix 3B), and counting cells ( appendix 3A)
NOTE: All solutions and equipment coming into contact with cells must be sterile and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Literature Cited

Literature Cited
   Albright, J.W. and Albright, J.F. 1983. Age‐associated impairment of murine natural killer activity. Proc. Natl. Acad. Sci. U.S.A. 80:6371‐6375.
   Blomberg, K., Hautala, R., Lövgren, J., Mukkala, V.‐M., Lindquist, C., and Akerman, K. 1996. Time‐resolved fluorometric assay for natural killer activity using target cells labelled with a fluorescence enhancing ligand. J. Immunol. Methods 193:199‐206.
   Herberman, R.B., Reinolds, C.W., and Ortaldo, J.R. 1986. Mechanism of cytotoxicity by natural killer (NK) cells. Annu. Rev. Immunol. 4:651‐664.
   Kimberley, M.G., Chapman, G., Marks, R., and Perry, R. 1986. A fluorescence NK assay using flow cytometry. J. Immunol. Methods 86:7‐13.
   Mullaney, P.F. and Dean, P.N. 1970. The small angle light scattering of biological cells. Theoretical considerations. Biophys. J. 10:764‐773.
   Papa, S., Vitale, M., Mariani, A.R., Roda, P., Facchini, A., and Manzoli, F.A. 1988. Natural killer function in flow cytometry. I. Evaluation of NK lytic activity on K562 cell line. J. Immunol. Methods 107:73‐78.
   Papa, S., Gregorini, A., Pascucci, E., Bartolucci, M., Rocchi, M.B.L., and Valentini, M. 1994. Inhibition of NK binding to K562 cells induced by MAb saturation of adhesion molecules on target membrane. Eur. J. Histochem. 38:83‐90.
   Segal, D.M. and Stephany, D.A. 1984. The measurement of specific cell‐cell interactions by dual‐parameter flow cytometry. Cytometry. 5:169‐174.
   Storkus, W.S., Balber, A.E., and Dawson, J.R. 1986. Quantitation and sorting of vitally stained natural killer cell‐target cell conjugates by dual beam flow cytometry. Cytometry 7:163‐169.
   Vitale, M., Rizzoli, R., Mariani, A.R., Neri, L.M., Facchini, A., and Papa, S. 1989. Evaluation of NK‐to‐target cell binding and evidence for T cell conjugates by flow cytometry. Cytotechnology. 2:59‐62.
   Vitale, M., Zamai, L., Papa, S., Mazzotti, G., Facchini, A., Monti, G., and Manzoli, F.A. 1992. Natural killer function in flow cytometry. III. Surface marker determination of K562‐conjugated lymphocytes by dual laser flow cytometry. J. Immunol. Methods. 149:189‐196.
   Zamai, L., Rana, R., Mazzotti, G., Centurione, L., Di Pietro, R., and Vitali, M. 1994. Lymphocyte binding to K562 cells: Effect of target cell irradiation and correlation with ICAM‐1 and LFA‐3 expression. Eur. J. Histochem. 38‐53‐60.
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