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Miltenyi Biotec Inc.
2303 Lindbergh Street
Auburn, CA 95602
USA
Phone: +1 800 FOR MACS
+1 530 888 8871
Fax: +1 530 888 8925
E-Mail: macs@miltenyibiotec.com
Web: www.miltenyibiotec.com
For immediate technical support, use our live chat.
Connect with usOrders:
Phone: +1 866 811 4466
Fax: +1 877 591 1060
E-Mail: orderdesk@miltenyibiotec.com
Technical support - Research products:
Phone: +1 800 FOR MACS
Fax: +1 530 745 2806
E-Mail: science@miltenyibiotec.com
Technical support - Clinical products:
Phone: +1 800 FOR MACS
Fax: +1 530 745 2806
E-Mail: science@miltenyibiotec.com
Miltenyi Biotec distribution:
As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
Our local employees are always happy to answer your questions. Highly trained and experienced teams in your country can provide quick, helpful, and comprehensive support.
Miltenyi Biotec Inc.
2303 Lindbergh Street
Auburn, CA 95602
USA
Phone: +1 800 FOR MACS
+1 530 888 8871
Fax: +1 530 888 8925
E-Mail: macs@miltenyibiotec.com
Web: www.miltenyibiotec.com
For immediate technical support, use our live chat. Connect with us
Orders
Phone: +1 866 811 4466
Fax: +1 877 591 1060
E-Mail: orderdesk@miltenyibiotec.com
Technical support - Research products
Phone: +1 800 FOR MACS
Fax: +1 530 745 2806
E-Mail: science@miltenyibiotec.com
Technical support - Clinical products
Phone: +1 800 FOR MACS
Fax: +1 530 745 2806
E-Mail: science@miltenyibiotec.com
As a global market leader with numerous subsidiaries and distributors, Miltenyi Biotec is committed to providing our customers around the world with the highest quality products. In addition to direct selling in more than 20 countries in North America, Europe and Asia/Pacific, Miltenyi Biotec also provides support for our customers through an extensive distributor network covering dozens of additional countries.
At a glance: Natural Killer (NK) cells in various tissues (PMID: 19234143, 16424180)
Frequencies: (PMID: 23034281, 23649470); Function: (PMID: 28261203)
Cell subset | Frequency | Markers | Function |
---|---|---|---|
CD11blowCD27low NK cells | 1.1-2.75% | CD27, CD11b | Found predominantly in bone marrow and very low frequency in all lymphoid and nonlymphoid organs |
CD11blowCD27high NK cells | 11.5-16.5% | CD27, CD11b | Found predominantly in bone marrow, lymph nodes and in a greater proportion in the liver Exhibit greater effector function and responsiveness to chemokines Produce larger amounts of cytokines Show decreased cytotoxicity Interact with dendritic cells |
CD11bhighCD27high NK cells | 23.2-49.3% | CD27, CD11b | Homogeneously distributed Exhibit greater effector function and responsiveness to chemokines Express higher levels of killer cell lectin-like receptor subfamily G, member 1 (KLRG1) Interact with dendritic cells |
CD11bhighCD27low NK cells | 31.4-58.3% | CD27, CD11b | Most abundant in blood, spleen, lung and liver Increased cytotoxicity against target cells |
Miltenyi Biotec has created dedicated application protocols to isolate and analyze NK cells
The main source for mouse NK cells is single-cell suspensions from spleen and lymph nodes, which are used for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis.
Dissociation can be fully automated using the gentleMACS™ Dissociators and dedicated tissue dissociation kits (e.g., Spleen Dissociation Kit, mouse). A special protocol for the preparation of single-cell suspensions from mouse spleen without enzymatic treatment can be downloaded from the Related Resources panel to the right. Alternatively, tissues can be dissociated manually. For details, see related chapters.
Magnetic cell separation
Starting material | Isolation strategy | Comments | Automation | Product |
---|---|---|---|---|
Single-cell suspensions from spleen and lymph nodes | Depletion of non-target cells | Isolation of all NK cells via depletion of all non-NK cells | Yes* | NK Cell Isolation Kit, mouse |
Positive selection of target cells | CD49b is expressed on NK cells and a small population of T cells (CD4+CD3+ TCRα/β+). CD49b is less mouse strain–specific than other NK cell markers (e.g. NK1.1) and is expressed by the most common inbred mouse strains | Yes* | CD49b (DX5) MicroBeads, mouse | |
Positive selection of target cells | NKp46 seems to be exclusively expressed on NK cells. Expression has been shown on BALB/c, SJL, CBA/CA, C57Bl/6, NOD, DBA/2, and B6.129 mice | Yes* | Anti-NKp46 MicroBead Kit, mouse | |
Sequential separation | Isolation of NK1.1+ iNKT cells via depletion of all NK1.1+ iNKT cells followed by enrichment via anti-NK1.1-APC and Anti-APC MicroBeads | Yes* | NK1.1+ iNKT Cell Isolation Kit, mouse | |
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. |
Before separation
Isolated NK cells
NK cell isolation from single-cell suspensions of spleen. NK cells were isolated from BALB/c mice using the NK Cell Isolation Kit, a LS Column, and a MidiMACS™ Separator. The cells were fluorescently stained with CD3-FITC, CD49b-APC, and Anti-NKp46-PE. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals, CD45-VioGreen™, and 4’,6-diamidino-2-phenylindole (DAPI) fluorescence.
Before separation
Isolated NK cells
NK cell isolation from single-cell suspensions of spleen. NK cells were isolated from C57BL/6 mice using the NK Cell Isolation Kit, a LS Column, and a MidiMACS™ Separator. The cells were fluorescently stained with CD3-FITC, CD49b-APC, and Anti-NK1.1-PE . Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals, CD45-VioGreen™, and 4’,6-diamidino-2-phenylindole (DAPI) fluorescence.
Unseparated fraction
After separation
Positive selection of NK cells. NKp46+ NK cells were isolated from BALB/c mouse spleen using the Anti-NKp46 MicroBead Kit, two consecutive MS Columns, and a MiniMACS™ Separator. Cells were fluorescently stained with CD3ε-APC and Anti-NKp46-PE and analyzed by flow cytometry using the MACSQuant Analyzer.
Distinct separation of CD49b (DX5)+ cells. Isolation of mouse NK cells from a single-cell suspension of mouse spleen using CD49b (DX5) MicroBeads, a MiniMACS Separator, and an MS Column.
MACS Cell Separation - Select the best (brochure)
Cap'n T Cell explains what makes MACS® Technology unique and superior and why it is the best choice for isolating your cells.
Basic NK lineage | Activating receptors, adhesion and effector functions | Inhibitory receptors | Transcription Factors | Ly49 | Secreted factors | Other interesting markers |
---|---|---|---|---|---|---|
NK1.1 | β-2 Integrins (CD11a-CD18, CD11b-CD18, CD11c-CD18) | LAIR-1 | CD278 (ICOS) | Ly49A | IFN-γ | CD25 |
CD127 | CD335 (NKp46) | CD94/NKG2A | EOMES | Ly49A/D | TNF-α | CD27 |
CD49b | DNAM-1 | SIGLEC-A | GATA3 | Ly-49C/F/I/H | IL-12 (p40/p70) | Tim-3 |
CD117 | 2B4/CD244 | KLRG1 | RORγ (t) | Ly-49C/I | GM-CSF | TIGIT |
CD11b | CD314 (NKG2D) | NKRP1-B, -D | T-bet | Ly-49E/F | CD279 (PD1) | |
CD122 | NKG2C | 2B4 (CD244) | TOX | Ly-49F | PDL2 | |
NKG2E | Helios | Ly-49G2 | PLZF | |||
CD25 | Icaros | Ly-49H | FcεR1γ | |||
TRAIL (CD253) | Ly-49I | FcεRIα | ||||
Granzyme B | Ly-49D | |||||
CD69 | Ly-49G | |||||
CD62L | ||||||
CD178 (FasL) | ||||||
CD107a (LAMP-1) | ||||||
Perforin | ||||||
LAG-3 |
Marker | Clone |
---|---|
Viability 405/520 Fixable Dye | - |
Anti-NK1.1-Pe-Vio770 | PK136 (soon REA632) |
CD11b-VioBlue | REA592 |
CD335 (NKp46)-FITC | REA815 |
CD27-APC | REA499 |
CD45-PE-Vio615 | REA737 |
Anti-KLRG1-PE | REA1016 |
CD3-APC-vio770 | REA641 |
CD19-APC-vio770 | REA749 |
REAfinity Recombinant Antibodies (brochure)
Spleen | Liver | Lung | Bone marrow | Uterus | |
CD3 | - | - | - | - | - |
CD49b (DX5) | + | - | + | + | -/+ |
CD49a | - | + | + | ||
CD27 | - | low | -/+ | - | |
NKp46 | + | + | + | + | + |
CD11b | + | -/low | high | -/+ | + |
TRAIL | # | ||||
Ly49 | + | -/low | high | + | high |
CD94/NKG2A | + | + | high | + | |
KLRG1 | low | + | + |
Sample preparation
Magnetic cell separation
MACS Cell Separation - Select the best (brochure)