Self-generating density gradients of Percoll provide a simple and rapid method that consistently enriches natural killer cells.
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.
Ravnik
S E
SE
Department of Biological Structure, University of Washington, School of Medicine, Seattle 98195.
Gage
S
S
Pollack
S B
SB
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
C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone