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Flow cytometric analysis of CCR7 (CD197) expression on CD4 and CD8-positive human peripheral lymphocytes. Human PBMC were stained with 20 ul/test of Biotin Rat Anti-Human CCR7 (CD197) (Cat. No. 552174) and FITC Mouse Anti-Human CD45RA (Cat. No. 555488). Secondary staining was carried out with PE Streptavidin (Cat. No. 554061). Bivariate dot plots are derived from the CD4-postive (based on staining with APC Mouse Anti-Human CD4, Cat. No. 555349, left panel) and CD8-positive (based on staining with APC Mouse Anti-Human CD8, Cat. No. 552174, right panel) lymphocyte gated populations.
BD Pharmingen™ Biotin Rat Anti-Human CCR7 (CD197)
Regulatory Status Legend
Any use of products other than the permitted use without the express written authorization of Becton, Dickinson and Company is strictly prohibited.
Preparation And Storage
Recommended Assay Procedures
The biotinylated 3D12 antibody can be used with a very bright second-step reagent, such as Streptavidin-phycoerythrin (Cat. No. 554061) for the immunofluorescent staining and flow cytometric analyses of human leukocytes and cell lines that express CCR7 (see Figure). We are unable to generate reproducible results using less bright reagent such as Streptavidin-FITC or -PerCP with this antibody. The use of Streptavidin-FITC or -PerCP is not recommended.
Recommendation: Chemokine receptors are know to internalize during manipulation resulting in low frequency expression. Immunophenotyping studies of chemokine receptors need to be performed on freshly collected whole blood (<24 Hrs). Incubation with the antibody should be done in the dark. Cellular manipulation, such as Ficoll separation, freezing, or exposure to cold temperatures prior to staining have been shown to cause a decrease in staining intensity and inconsistent results.
A multiple-step staining procedure is strongly recommended to amplify immunofluorescent signals in the flow cytometric analysis of human CCR7 expression. Purified Rat Anti-Human CD197 (CCR7) (Cat. No. 552175) and a 3-step staining procedure is recommended for detecting low frequency expression.
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
The monoclonal antibody 3D12 reacts with the human CC chemokine receptor, CCR7. CCR7 (previously known as BLR-2, EBI-1 and CMKBR7), a seven-transmembrane, G-protein-coupled receptor, is the specific receptor for CC chemokines, MIP-3β/Exodus 3/ELC/ CCL19 and 6Ckine/Exodus 2/SLC/TCA4/CCL21. It has been shown that CCR7 mRNA is expressed mainly in lymphoid tissues including spleen, lymph nodes and tonsil. CCR7 mRNA was also detected in peripheral T and B lymphocytes, in bone marrow and cord blood CD34-positive cells and mature dendritic cells. The human CCR7 gene, unlike other CC chemokine receptor genes, has been mapped to chromosome 17q12. The immunogen used to generate 3D12 hybridoma was the N-terminus as well as parts of the second extracellular loop of human CCR7 protein. The monoclonal antibody 3D12 recognizes an epitope mapping to the N-terminus of human CCR7.
Development References (15)
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Schweickart VL, Raport CJ, Godiska R, et al. Cloning of human and mouse EBI1, a lymphoid-specific G-protein-coupled receptor encoded on human chromosome 17q12-q21.2. Genomics. 1994; 23(3):643-650. (Biology). View Reference
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Yanagihara S, Komura E, Nagafune J, Watarai H, Yamaguchi Y. EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J Immunol. 1998; 161(6):3096-3102. (Biology). View Reference
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Yoshida R, Imai T, Hieshima K, et al. Molecular cloning of a novel human CC chemokine EBI1-ligand chemokine that is a specific functional ligand for EBI1, CCR7. J Biol Chem. 1997; 272(21):13803-13809. (Biology). View Reference
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Yoshida R, Nagira M, Imai T, et al. EBI1-ligand chemokine (ELC) attracts a broad spectrum of lymphocytes: activated T cells strongly up-regulate CCR7 and efficiently migrate toward ELC. Int Immunol. 1998; 10(7):901-910. (Biology). View Reference
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Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.