Human natural killer cells: a convenient purification procedure and the influence of cryopreservation on cytotoxic activity

J Immunol Methods. 1993 Sep 27;165(1):21-30. doi: 10.1016/0022-1759(93)90102-d.


The recognition of natural killer cells as a lymphoid subpopulation with a distinct set of surface markers has led to the development of a variety of antibody-based purification methods. In this paper we describe a rapid, three-step negative selection protocol for the purification of human natural killer (NK) cells from the mononuclear cell fraction, which is obtained by the centrifugation of peripheral blood on Ficoll-Paque. Subsequently, monocytes and B lymphocytes are removed by adherence to nylon wool and T lymphocytes by panning with anti-CD3. With this procedure, CD3-, CD16/56+ NK cells are purified about five-fold, from 12 +/- 3% in the starting population to a final purity of 61 +/- 11%. A further increase to > or = 70% is obtained, if an extra Ficoll centrifugation step is included. The recovery of NK cells (50%) is significantly higher than is usually achieved by previously described procedures. Furthermore, we show that activation of cytotoxicity, with concomitant changes in target specificity, occurs when frozen/thawed NK effector cells are kept in culture in order to regain their pre-freezing cytotoxicity levels.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Separation / methods
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Cryopreservation*
  • Cytotoxicity, Immunologic
  • Flow Cytometry
  • Humans
  • Killer Cells, Natural / cytology*
  • Killer Cells, Natural / immunology
  • Lymphocyte Activation
  • Tumor Cells, Cultured