Technical Note: R&D Systems MagCellect Human & Mouse Natural Killer Cell Isolation Kits

Natural killer (NK) cells are lymphocytes of the innate immune system that function as both cytolytic effectors and regulators of immune responses. NK cells develop from hematopoietic stem cells primarily in the bone marrow and are educated to become self-tolerant by recognition of self MHC class I molecules. Mature NK cells migrate to the liver and peripheral lymphoid organs, including the spleen and lymph nodes. They are activated upon detection of abnormalities in target cells including loss or down-regulation of MHC class I expression, or upregula­tion of stress-induced ligands that occurs in response to infection or neoplastic transformation.

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Enrichment of NK Cells from Peripheral Blood Mononuclear Cells using the MagCellect Human NK Cell Isolation Kit. The MagCellect Human NK Cell Isolation Kit (Catalog # MAGH109) was used to enrich for NK cells from a peripheral blood mononuclear cell suspension. Cells were double-stained with an APC-conjugated Anti-Human CD3 Monoclonal Antibody (Catalog # FAB100A) and PE-conjugated Anti-Human NCAM-1/CD56 Monoclonal Antibody (Catalog # FAB2408P) before (A) and after (B) enrichment. CD56dim cells are shown in green and CD56bright cells are shown in red. (C) Corresponding histograms of the CD56 staining before and after selection are shown.

R&D Systems has developed two MagCellect Kits for mouse and human NK cell selection. Both kits are designed to isolate NK cells using a negative selection protocol to remove undesired cells from a mononuclear cell suspension. Unlike positive selection kits, the MagCellect Kits leave the NK cell populations untouched, reducing the risk that the cells will be altered by the selection process. The typical purity of the recovered NK cells ranges between 70-80% for the mouse kit and 80-90% for the human kit.

The effectiveness of the negative selection protocol for NK cell isolation was determined by characterizing the phenotypes and the functional capabilities of the cells following isolation. These experiments demonstrated that the isolated cells express a variety of NK cell-specific markers including CD56, NKp46, NKp80, NKp30, NKG2D, KIR3DL1, and NTB-A in human, and NKp46, NKG2D, and CD49b in mouse (see data sheets online). In addition, cells isolated with the human NK Cell Isolation Kit were shown to undergo target cell-induced degranulation (middle panel), and to express NKp46 and Granzyme B by immunohistochemisty (bottom panel). These experiments confirmed that NK cells isolated with the MagCellect Kits express the appropriate markers and remain biologically active.

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Detection of Target Cell-induced NK Cell Degranulation following Isolation using the MagCellect Human NK Cell Isolation Kit. Human peripheral blood natural killer (NK) cells were isolated using the MagCellect Human NK Cell Isolation Kit (Catalog # MAGH109). Isolated cells were incubated alone (A), or with K562 human erythroleukemia cells (B), at an effector to target ratio of 2:1 for 3 hours. NK cell degranulation, as indicated by translocation of LAMP1/CD107a to the cell membrane, was analyzed using an APC-conjugated Anti-Human LAMP1/CD107a Monoclonal Antibody (Catalog # IC4800A).

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Detection of NKp46 and Granzyme B Expression in Human NK Cells Isolated using the MagCellect Human NK Cell Isolation Kit. Human peripheral blood natural killer (NK) cells were isolated using the MagCellect Human NK Cell Isolation Kit (Catalog # MAGH109). NKp46 was detected in isolated cells using a Goat Anti-Human NKp46 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1850) followed by staining with a NorthernLights™ 493-conjugated Anti-Goat Secondary Antibody (Catalog # NL003; green). Granzyme B was detected using a Mouse Anti-Human Granzyme B Monoclonal Antibody (Catalog # MAB2906) followed by staining with a NorthernLights 557-conjugated Anti-Mouse Secondary Antibody (Catalog # NL007; red). Nuclei were counterstained with DAPI (blue).