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Multiparameter flow cytometric analysis of CCR7 (CD197) on Human peripheral blood leukocytes and lymphocytes. Whole blood was stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 (Cat. No. 564724), FITC Mouse Anti-Human CD8 (Cat. No. 561947/555366/557085), BD Horizon™ BV421 Mouse Anti-Human CD45RA (Cat. No. 562885) antibodies, and either PE-Cy7 Mouse IgG1, κ Isotype Control (Cat. No. 565573) or PE-Cy7 Mouse Anti-Human CCR7 (CD197) antibody (Cat. No. 567313/567314). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). Flow cytometry and data analysis were performed using a BD FACS LSRFortessa™ X20 Flow Cytometer and FlowJo™ software. Plots 1 and 2: Bivariate pseudocolor density plots showing Ig isotype control or CCR7 (CD197) staining versus side light-scatter (SSC-A) were derived from gated events with the forward and side light-scatter characteristics of viable leucocytes. Plot 3: Bivariate pseudocolor density plot showing CCR7 (CD197) staining versus CD45RA was derived from CD4-positive T cell gated events with the forward and side light-scatter characteristics of intact lymphocytes. Please note that we have observed considerable donor-to-donor variation in the proportion of CCR7 (CD197)-negative CD4-positive lymphocytes. Plot 4: Bivariate pseudocolor density plot showing CCR7 (CD197) staining versus CD45RA was derived from CD8-positive T cell gated events with the forward and side light-scatter characteristics of intact lymphocytes.
BD Pharmingen™ PE-Cy7 Mouse Anti-Human CCR7 (CD197)
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Preparation And Storage
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cell and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
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- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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).
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- Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
- PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), which is excited by 488-nm light and serves as an energy donor, coupled to the cyanine dye Cy7, which acts as an energy acceptor and fluoresces maximally at 780 nm. PE-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher. Although every effort is made to minimize the lot-to-lot variation in the efficiency of the fluorochrome energy transfer, differences in the residual emission from PE may be observed. Therefore, we recommend that individual compensation controls be performed for every PE-Cy7 conjugate. PE-Cy7 is optimized for use with a single argon ion laser emitting 488-nm light, and there is no significant overlap between PE-Cy7 and FITC emission spectra. When using dual-laser cytometers, which may directly excite both PE and Cy7, we recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
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The 2-L1-A monoclonal antibody specifically binds to the human CC chemokine receptor CCR7, also known as CD197, on the cell surface. CCR7 (previously known as BLR2, EBI1 and CMKBR7) is a seven-transmembrane, G-protein-coupled receptor specific for two CC chemokines: CCL19 (also known as MIP-3β, Exodus-3, and ELC) and CCL21 (also known as 6Ckine, Exodus-2 SLC, TCA4, and SCYA21). CCR7 mRNA is expressed mainly in lymphoid tissues including the spleen, lymph nodes and tonsil, in bone marrow, and on peripheral T and B lymphocytes, cord blood CD34-positive cells, and mature dendritic cells. In response to its cognate chemokines, CCR7 (CD197) mediates homing of leucocytes to secondary lymphoid tissues. Differential CCR7 (CD197) expression can be used to distinguish naive, central memory, and effector memory T cell subsets. The human CCR7 gene, unlike other CC chemokine receptor genes, has been mapped to chromosome 17 (region 17q12). Because the extracellular region of CCR2 (CD192) has significant sequence homology with CCR7 (CD197), BD Biosciences has confirmed that mAb 2-L1-A does not cross-react with CCR2 on the surface of transfected cells.
Development References (8)
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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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