I have purified NK cells from human peripheral blood mononuclear cells, using "negative" selection in an attempt to minimize non-specific activation. The PBMC were labelled with a cocktail of mouse monoclonal antibodies: CD3, CD5, CD13, CD14, CD19, and anti-glycophorin A. They were then subjected to two cycles of depletion with goat anti-mouse IgG coated Dynabeads (using bead to cell ratios of 5:1 the first time and 10:1the second time). This enriched population was then labelled with sheep anti-mouse IgG-FITC and sorted on the flow cytometer for the FITC-negative cells. This gave a population >99% positive for CD56 and/or CD16, and negative for CD4. The procedure is time consuming (and expensive) but does yield pure, healthy, and functional NK cells. Malcolm King Dept Clinical Immunology Royal North Shore Hospital St Leonards NSW 2065 Australia >>> "Michael Dustin " <dustin@saturn.med.nyu.edu> 02/15/01 04:05pm >>> Hi, Does anyone have experience with sorting NK cell populations from peripheral blood? The entire population is 5-10% and there are lots of markers that split this population that would be useful. If so, are the sorted cells functional in killing? Thanks, Mike Michael L. Dustin, Ph.D. Irene Diamond Associate Professor of Immunology Program in Molecular Pathogenesis Skirball Institute of Molecular Medicine Department of Pathology New York University School of Medicine 540 First Avenue, 5th Floor New York, NY 10016 Office- (212) 263-3207 Lab- (212) 263-3208 Project assistant- (212) 263-6282 FAX- (212)-263-5711 dustin@saturn.med.nyu.edu
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