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Highly pure, viable and functional intact human Natural Killer (NK) cells isolated in a single isolation step using Dynabeads® FlowCompTM Technology
Author(s) -
Schjetne Karoline Western,
Kullman Anette,
Møller Anne Sophie,
Kierulf Bente,
Lycke kirsten,
Karlsson Malin,
Neurauter Axl
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.676.8
Subject(s) - flow cytometry , cytotoxicity , peripheral blood mononuclear cell , biology , k562 cells , antibody , immune system , cd3 , microbiology and biotechnology , chemistry , cell , immunology , cd8 , in vitro , biochemistry
BACKGROUND NK cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. In humans, NK cell express CD56. However, this surface marker is not exclusive for NK cells and separation technology focusing on this marker will potentially result in contamination of non‐NK cells or involve several isolation steps. Here we present a single step, positive isolation protocol for human NK cells. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from healthy individuals. NK‐specific antibody conjugated to a modified biotin was added and incubated at 2–8 °C. In the next step modified streptavidin coated Dynabeads were added and placed in the magnet after incubation at room temperature. After resuspension with FlowComp release buffer the bead‐free cells were analyzed on flow cytometry and tested in cytotoxicity assays. RESULTS: Highly pure human CD56 + CD3 − NK cells were isolated from healthy blood donors using Dynabeads® FlowComp™ Human NK Cell Kit. Using the single step positive isolation protocol, very few CD3 + NKT/T cells were present after isolation and viability was >95%. Isolated NK cells were used in functional assays where LAMP‐1 expression was assayed by flow cytometry and where cytotoxicity was measured in a 51 Cr release assay using K562 as target cells. We demonstrate that isolated NK cells were activated and kill target cells.