Human natural killer cells: a convenient purification procedure and the influence of cryopreservation on cytotoxic activity.
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.
Voshol
H
H
Utrecht University, Bijvoet Center, Department of Bio-Organic Chemistry, Netherlands.
Dullens
H F
HF
Den Otter
W
W
Vliegenthart
J F
JF
D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D015925:Cryopreservation; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology* / Q000276:immunology; D008213:Lymphocyte Activation; D014407:Tumor Cells, Cultured