TY - JOUR
T1 - Self-generating density gradients of Percoll provide a simple and rapid method that consistently enriches natural killer cells
AU - Ravnik, Stuart E.
AU - Gage, Sarah
AU - Pollack, Sylvia B.
N1 - Funding Information:
S.R. was supported by the Molecular and Cellular Training Program, NRSA Grant no. 5-T32-GM0720. Further support for this project was from NIH-CA-32553.
PY - 1988/6/13
Y1 - 1988/6/13
N2 - 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 × 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.
AB - 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 × 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.
KW - Cytotoxicity
KW - Natural killer cell
KW - Percoll
KW - Self-generating gradient
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U2 - 10.1016/0022-1759(88)90099-3
DO - 10.1016/0022-1759(88)90099-3
M3 - Article
C2 - 2837514
AN - SCOPUS:0023900405
SN - 0022-1759
VL - 110
SP - 161
EP - 168
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 2
ER -