Product Information

Contents: Alexa Fluor® 700 anti-mouse CCR7 (CD197, EBI-1)
Catalog Number: 56-1971
Sizes: 25 ug, 100 ug
Formulation: Phosphate buffer pH 7.2,
150 mM NaCl, 0.09% NaN₃, 0.1% gelatin
Storage Conditions: Store at 4°C.
DO NOT FREEZE.
LIGHT-SENSITIVE MATERIAL.
Clone: 4B12
Isotype: Rat IgG2a, κ

Staining of C57Bl/6 splenocytes with FITC anti-mouse CD3e (145-2C11) (cat. 11-0031) and 0.5 μg of Alexa Fluor® 700 Rat IgG2a Iso Cntrl (cat. 56-4321) (left) or 0.5 μg of Alexa Fluor® 700 anti-mouse CCR7 (4B12) (right). 4B12 staining was carried out at 37°C for 0.5 hours and Fc receptor binding was blocked by staining cells with 5 µg/million cells anti-mouse CD16/32 (cat. 14-0161). Cells in the lymphocyte gate were used for analysis.

Available Formats of This Product
Cat. No.	Format	Excite (nm)	Emit (nm)	Reported Applications
12-1971	Phycoerythrin (PE) anti-mouse CCR7 (CD197, EBI-1)	488	575	FC
13-1971	Biotin anti-mouse CCR7 (CD197, EBI-1)	N/A	N/A	FC
14-1971	Affinity Purified anti-mouse CCR7 (CD197, EBI-1)	N/A	N/A	FC IP
16-1971	Functional Grade* Purified anti-mouse CCR7 (CD197, EBI-1)	N/A	N/A	FC IP
17-1971	Allophycocyanin (APC) anti-mouse CCR7 (CD197, EBI-1)	633	660	FC
25-1971	Phycoerythrin-Cy7 (PE-Cy7) anti-mouse CCR7 (CD197, EBI-1)	488	760	FC
45-1971	PerCP-Cy5.5 anti-mouse CCR7 (CD197, EBI-1)	488	690	FC
56-1971	Alexa Fluor® 700 anti-mouse CCR7 (CD197, EBI-1) (APC-Cy5.5 Equivalent)	633	723	FC
*Functional Grade™ (FG™) Purified: Azide-free, sterile-filtered, and endotoxin < 0.001 ng/µg (unless otherwise noted). *Functional Grade™ (FG™) Biotin: Azide-free, sterile-filtered, and endotoxin < 0.05 ng/µg. Purified: Contains azide, not sterile-filtered, and not endotoxin tested.

Questions? Please consult our answers to frequently asked questions at http://www.ebioscience.com/faq.

Description

Please see our website for continuous updates regarding the use of this product.

The 4B12 monoclonal antibody reacts with mouse CCR7, also known as EBI-1 and CD197. For optimal visualization of CCR7 expression on different cell types it is necessary to use multi-color staining to discriminate different cell subsets (please see Technical Data Sheets for recommended concentration for individual formats). To address specificity, the staining profile of 4B12 has been compared to a polyclonal antibody generated against a CCR7 peptide (Bjorkdahl et al). This analysis confirms that the polyclonal antibody and 4B12 stain similar populations of cells. Furthermore, 4B12 stains mouse CCR7-GFP fusion protein-transfected RBL cells (see data in cat. 14-1971).

CCR7 is a chemokine receptor for the chemokines CCL19 (CKß11, ELC, MIP3ß, Scya19, Exodus-3) and CCL21 (CKß9, SLC, MIP2ß, Scya21, Exodus-2). In recent years, the role of chemokines in directing the migration of lymphocytes has been well-characterized. One of the most important mediators of homeostatic trafficking of naïve T cells to secondary lymphoid organs (SLO) is the chemokine receptor CCR7. Binding of its ligands, CCL19 and CCL21, mediates the transendothelial migration of T cells across high endothelial venules into SLO. It has also been demonstrated that CCR7 plays a role in the localization of dendritic cells and B cells during an immune response.

In addition to its significant role in the chemotaxis of lymphocytes, human CCR7 has also been recognized as a marker for a distinct subset of memory T cells, the central memory (TCM) population. These cells are characterized by the expression of CCR7 and CD62L and reside within peripheral lymphoid organs.

CCR7 also plays a role in thymocyte development and its deficiency leads to disturbed thymic architecture, aberrant T cell development, and limited thymocyte expansion.

Applications Reported

For research use only, not for diagnostic or therapeutic use. This 4B12 antibody has been reported for use in flow cytometric analysis.

The Alexa Fluor® 700 emits at 723 nm and can be excited with the He-Ne 633 laser. Most instruments will require a 685 LP mirror and 710/20 filter. Please make sure that your instrument is capable of detecting this fluorochome.

Applications Tested

The 4B12 antibody has been tested by flow cytometric analysis of C57BL/6, Balb/C and SJL/J splenocytes and thymocytes.

Important: Staining with the 4B12 monoclonal antibody requires different conditions than typically used for surface-antigen staining. Please use the protocol below. Moreover, we have found that staining at 37°C, rather than 4°C, results in brighter 4B12 staining, as well as better resolution between positive and negative populations. Please see the data above which demonstrates a comparison of staining at 4°C and 37°C. Staining with 4B12 at 37°C is not expected to interfere with co-staining other antigens, however this should be evaluated for individual experiments.

1. Prepare cell suspension as normal and block FcγIIIR/FcγIIR with 5 μg/million cells purified anti-mouse CD16/32 (cat. 14-0161) for 15 minutes on ice. If red blood cell lysis is carried out as part of cell preparation, ensure that fixatives are not present in the red blood cell lysis solution as this will eliminate 4B12 staining.

Protocol for RBC Lysis of Mouse Spleen

2. Without washing, add 1 μg/million cells 4B12 and incubate in a 37°C waterbath or at 4°C (please see notes above) for 0.5 hours.

3. Wash cells 1X with 3 mls of Flow Cytometry Staining Buffer (cat. 00-4222) and decant supernatant.

4. Analyze cells on flow cytometer or proceed with secondary staining on ice as normal.

Note: Co-staining mouse CCR7 with the 4B12 antibody and the CCR7 ligand CCL19-Fc (cat. 14-1972) may be difficult due to different binding conditions required for the antibody versus the ligand, and steric hindrance which may prevent co-staining of 4B12 and CCL19-Fc. Cross-blocking experiments have demonstrated that 4B12 binding is able to prevent the detectable binding of CCL19-Fc, however not the opposite. Furthermore, the correlation between 4B12 and CCL19-Fc staining may be difficult to predict due to the presence of unknown CCL19-Fc receptors in addition to CCR7.

References

Ritter, U, Wiede, F, Mielenz, D, Kiafard, Z, Zwirner, J, and Korner H. 2004. Analysis of the CCR7 expression on murine bone arrow-derived and spleen dendritic cells. Journal of Leukocyte Biology. 76: 472-476. (4B12, FC, Pubmed)

Bjorkdahl O, Barber KA, Brett SJ, Daly MG, Plumpton C, Elshourbagy NA, Tite JP, Thomsen LL. 2003. Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues. Immunology. 110(2):170-9.

Waldner H, Sobel RA, et al. 2006. The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells. J Immunol. 176(9):5455-62. (4B12, FC, PubMed)

Ohl L, Mohaupt M, Czeloth N, Hintzen G, Kiafard Z, Zwirner J, Blankenstein T, Henning G, Forster R. 2004. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity. 21(2):279-88. (4B12, FC, Pubmed)

Related Products

Cat. 56-4321 Alexa Fluor® 700 Rat IgG2a Isotype Control (APC-Cy5.5 Equivalent)

Alexa Fluor® and Pacific Blue® are registered trademarks of and licensed under patents assigned to Molecular Probes, Inc. for research use only. This product is subject to an agreement between Molecular Probes, Inc. and eBioscience, and the manufacture, use, sale or import of this product may be subject to one or more U.S. patents, pending applications and corresponding foreign equivalents, owned by Molecular Probes, Inc. (a wholly owned subsidiary of Invitrogen Corp). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product for life science research or as an ASR. The buyer cannot use this product for manufacturing or for any other screening (specifically including use in combination with microarrays or High Content Screening) or testing purpose, other than as an ASR. For information on purchasing a license to this product for purposes other than life science research or use as an ASR, contact Molecular Probes, Inc.