Tuesday, December 8, 2009

New Human Natural Killer Cell purification kit

We have developed two new kits for the isolation of untouched Natural Killer cells from both mouse and human preparations that achieves levels of purity as high as 95%. Undesired populations are negatively depleted using a cocktail of monoclonal antibodies that specifically react with non-NK cell populations. These cell types are then tagged with magnetic beads and separated from the desired NK cells by magnetic force. A typical isolation is achieved in 45 minutes.

Introducing the new Human NK Cell Isolation Kit [Catalog# MAGH109]:



Two examples of enrichment of NK cells from human PBMC using MagCellect NK
cell enrichment kit (R&D Systems MAGH109). Cells before and after enrichment
were double-stained with APC-conjugated anti human CD3 (R&D Systems FAB100A) and PE-conjugated anti human CD56 (R&D Systems FAB2408) to specifically label the NK cell populations. Panel A, Isolation of NK cells from a PBMC prep consisting of mostly CD56dim cells. Panel B, enrichment of both CD56dim and CD56bright from a different PBMC preparation; CD56dim NK cells are shown in green and CD56bright NK cells are shown in blue.

Phenotypic characterization of MagCellect-isolated NK cells

We have used the most recent and established markers and techniques to extensively characterize the isolated NK cells. We show here that the highly pure NK cell populations (both mouse and human) express NK cell-specific markers (i.e., NKp46, Nkp80, NKp30, CD56, NKG2D, KIR3DL1 and NTB-A)

List of R&D Systems APC- or PE-conjugated antibodies used in the phenotypic characterization of isolated NK cells:

Marker- Catalog # (click on the cat# to see more info)
NKp46 - FAB1850A
NKp44 - FAB22491A
NKp80 - FAB1900A
NKp30 - FAB1849P
CD56 - FAB2408A
NTB-A - FAB19081A
NKG2A - FAB1059A
Functionality of MagCellect-isolated NK cells

1) Isolated human NK cells were probed in a degranulation assay by measuring expression of CD107a (LAMP-1) in a flow cytometric assay after stimulation of isolated NK cells with myelogenous leukaemia K562 cells.

Enriched human NK cells were functional as demonstrated with a CD107adegranulation potential assay. NK cells were incubated with HLA class I- K562 target cells at an Effector/Target (E/T) ratio of 2:1 for 3 hours (Ravet, S. et al (2007) Blood 109:4296). Cells were then stained with APC-labeled anti-human CD107a/LAMP1 (R&D Systems Catalog #IC4800A) and analyzed by flow cytometry.

2) Interferon gamma induction on isolated NK cells was assayed

Immobilized Ms x hNKp30 (R&D Systems Inc. catalog #MAB1849) induces IFNγ secretion in IL-2 activated human NK cells. Isotype control did not induce IFNγ secretion. Human peripheral blood NK cells were isolated using MagCellect Human NK cell isolation kit (R&D Systems Inc. catalog #MAGH109). The amount of IFNγ secretion was measured using the human IFNγ kitQuantikine (R&D Systems Inc. catalog #DIF50).

Granzyme expression in intact isolated NK cells

MagCellect (MAGH109)-Isolated NK cells were stained with anti-human NKp46 (Catalog # AF1850) followed by NL493-coupled anti-goat IgG (Catalog # NL003; green), and with anti-human Granzyme B (Catalog # MAB2906) followed by NL557-coupled anti-mouse IgG (Catalog # NL007; red) in the dark. Nuclei were stained with Dapi (blue).
We tested the efficacy of our new MagCellect NK cell kits by comparison to other systems in the market, with typically better or similar results. But unlike other commercially available kits that require a costly set of magnets and supplies (expensive magnets and columns), our kits were developed to work with different kind of commercially available magnets and systems, providing more flexibility, simplicity and cost-efficacy.
We realize that some people already invested large amounts of money in costly magnets and supplies from our competitors, so we developed our MagCellect kits to be able to work with optimal performance using different type of magnets and supplies.

To see how our kits compare to other commercially available isolations systems, and how you can effectively use MagCellect NK Isolation kits with other suppliers magnets and/or column, check out the data presented here.

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