title: Self-generating density gradients of Percoll provide a simple and rapid method that consistently enriches natural killer cells.
abstract: The separation or enrichment of natural killer (NK) cells from the heterogeneous cell populations in murine spleen or bone marrow is a vital step for the study of NK cells. We report in this study a simple and rapid method for the enrichment of NK cells from B cell-depleted spleen cells, using a self-generating density gradient of polyvinyl pyrrolidone-coated silica (Percoll). Nylon wool-passed spleen cells are suspended in Percoll that is isotonic and isosmotic with mouse blood at a density of 1.087 g/ml and ultracentrifuged at 30,000 x g for 10 min. This method consistently enriches for NK cell cytotoxic activity, in spleen cells of both unstimulated and interferon-stimulated mice, as measured in the chromium release assay. There is a concomitant enrichment for cells bearing the NK marker asialo GM-1 and depletion of L3T4 or Lyt-2-bearing T cells. In contrast to discontinuous, step-wise gradients, the self-generating Percoll gradient, which relies on the intrinsic property of Percoll to form a continuous density gradient, appears to provide the cells with a physiological environment both before and during the centrifugation step.
authors.affiliation: Department of Biological Structure, University of Washington, School of Medicine, Seattle 98195.
authors.forename: S E
authors.initials: SE
authors.lastname: Ravnik
authors.forename: S
authors.initials: S
authors.lastname: Gage
authors.forename: S B
authors.initials: SB
authors.lastname: Pollack
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003601:Cytotoxicity Tests, Immunologic; D005260:Female; D007694:Killer Cells, Natural / Q000145:classification / Q000166:cytology / Q000276:immunology*; D007958:Leukocyte Count; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D011205:Povidone; D012822:Silicon Dioxide; D013154:Spleen / Q000166:cytology
chemical_list: C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone