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BB515 Rat Anti-Human CCR7 (CD197)
BB515 Rat Anti-Human CCR7 (CD197)

Multicolor flow cytometric analysis of CCR7 (CD197) expression on CD4+ and CD8+ human peripheral blood lymphocytes: staining comparisons between BD Horizon BB515- and FITC- conjugated antibodies. Human whole blood was stained with PerCP-Cy™5.5 Mouse Anti-Human CD4 (Cat. No. 560650), Alexa Fluor® 647 Mouse Anti-Human CD8 (Cat. No. 557708), BD Horizon™ BV421 Mouse Anti-Human CD45RA (Cat. No. 562885), and either BD Horizon™ BB515 Rat Anti-Human CCR7 (CD197) (Cat. No. 565869/565870; Top Plots), or FITC Rat Anti-Human CCR7 (CD197) (Cat. No. 560548/561675; Bottom Plots) antibodies. Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The two-color flow cytometric dot plots showing the correlated expression of CD45RA versus CCR7 (CD197) were derived from CD4+ (Left Plots) or CD8+ (Right Plots) gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.

Multicolor flow cytometric analysis of CCR7 (CD197) expression on CD4+ and CD8+ human peripheral blood lymphocytes: staining comparisons between BD Horizon BB515- and FITC- conjugated antibodies. Human whole blood was stained with PerCP-Cy™5.5 Mouse Anti-Human CD4 (Cat. No. 560650), Alexa Fluor® 647 Mouse Anti-Human CD8 (Cat. No. 557708), BD Horizon™ BV421 Mouse Anti-Human CD45RA (Cat. No. 562885), and either BD Horizon™ BB515 Rat Anti-Human CCR7 (CD197) (Cat. No. 565869/565870; Top Plots), or FITC Rat Anti-Human CCR7 (CD197) (Cat. No. 560548/561675; Bottom Plots) antibodies. Erythrocytes were lysed with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899). The two-color flow cytometric dot plots showing the correlated expression of CD45RA versus CCR7 (CD197) were derived from CD4+ (Left Plots) or CD8+ (Right Plots) gated events with the forward and side light-scatter characteristics of viable lymphocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ Cell Analyzer System.

Product Details
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BD Horizon™
CCR7, BLR-2, EBI-1, CMKBR7
Human (QC Testing)
Rat IgG2a, κ
Human CCR7 protein
Flow cytometry (Routinely Tested)
5 µl
1236
AB_2744305
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with BD Horizon™ BB515 under optimum conditions and unconjugated antibody was removed.

Recommended Assay Procedures

BD™ CompBeads can be used as surrogates to assess fluorescence spillover (Compensation).  When fluorochrome conjugated antibodies are bound to 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 cells and CompBead to ensure that BD Comp beads are appropriate for your specific cellular application.

For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime two or more BD Horizon Brilliant dyes are used in the same experiment.  Fluorescent dye interactions may cause staining artifacts which may affect data interpretation.  The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions.  More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385).

For optimal results, it is recommended to perform 2 washes after staining with antibodies. Cells may be prepared, stained with antibodies and washed twice with wash buffer per established protocols for immunofluorescence staining, prior to acquisition on a flow cytometer. Performing fewer than the recommended wash steps may lead to increased spread of the negative population.

Product Notices

  1. 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).
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. 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.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. Diagnostic uses require a separate license.
  6. Cy is a trademark of GE Healthcare.
  7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  8. BD Horizon Brilliant Stain Buffer is covered by one or more of the following US patents: 8,110,673; 8,158,444; 8,575,303; 8,354,239.
  9. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
565869 Rev. 3
Antibody Details
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3D12

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.

The antibody was conjugated to BD Horizon BB515 which is part of the BD Horizon Brilliant™ Blue family of dyes. With an Ex Max near 490 nm and an Em Max near 515 nm, BD Horizon BB515 can be excited by the blue laser (488 nm) laser and detected with a 530/30 nm filter. This dye has been exclusively developed by BD Biosciences and is up to seven times brighter than FITC with less spillover into the PE channel. Due to similar excitation and emission properties, BB515, FITC, and Alexa Fluor® 488 cannot be used simultaneously. It is not recommended to use BB515 in cocktails that include Streptavidin conjugates as it may cause high background.

565869 Rev. 3
Format Details
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BB515
The BD Horizon Brilliant™ Blue 515 (BB515) dye is part of the BD Horizon Brilliant™ Blue family of dyes. This dye is a polymer fluorochrome with an excitation maximum (Ex Max) at 490-nm and an emission maximum (Em Max) of 515-nm. Driven by BD innovation, BB515 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 520-nm (e.g., 530/30-nm). BB515 reagents are significantly brighter than equivalent FITC or Alexa Fluor™ 488 reagents with less spillover into the PE detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
BB515
Blue 488 nm
490 nm
515 nm
565869 Rev.3
Citations & References
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Development References (15)

  1. Birkenbach M, Josefsen K, Yalamanchili R, Lenoir G, Kieff E. Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors. Nature. 1993; 67(4):2209-2220. (Biology). View Reference
  2. Britschgi MR, Link A, Lissandrin TK, Luther SA. Dynamic modulation of CCR7 expression and function on naive T lymphocytes in vivo. J Immunol. 2008; 181(11):7681-7688. (Biology). View Reference
  3. Burgstahler R, Kempkes B, Steube K, Lipp M. Expression of the chemokine receptor BLR2/EBI1 is specifically transactivated by Epstein-Barr virus nuclear antigen 2. Biochem Biophys Res Commun. 1995; 215(2):737-743. (Biology). View Reference
  4. Forster R, Davalos-Misslitz AC, Rot A. CCR7 and its ligands: balancing immunity and tolerance. Nat Rev Immunol. 2008; 8(5):362-371. (Biology). View Reference
  5. Kim CH, Pelus LM, White JR, Broxmeyer HE. Macrophage-inflammatory protein-3 beta/EBI1-ligand chemokine/CK beta-11, a CC chemokine, is a chemoattractant with a specificity for macrophage progenitors among myeloid progenitor cells. J Immunol. 1998; 161(5):2580-2585. (Biology). View Reference
  6. Kurobe, H., Liu, et al. CCR7-dependent cortex-to-medulla migration of positively selected thymocytes is essential for establishing central tolerance. Immunity. 2006; 24(2):165-177. (Biology). View Reference
  7. Lipp M, Burgstahler R, Muller G, et al. Functional organization of secondary lymphoid organs by the chemokine system. Curr Top Microbiol Immunol. 2000; 251:173-179. (Immunogen: Functional assay, Inhibition). View Reference
  8. Ohl L, Mohaupt M, Czeloth N, et al. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity. 2004; 21(2):279-288. (Biology). View Reference
  9. Ritter U, Wiede F, Mielenz D, Kiafard Z, Zwirner J, Korner H. Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells.. J Leukoc Biol. 2004; 76(2):472-476. (Biology). View Reference
  10. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature. 1999; 401(6754):708-712. (Clone-specific: Flow cytometry). View Reference
  11. 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
  12. 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
  13. 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
  14. 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
  15. Yoshida R, Nagira M, Kitaura M, Imagawa N, Imai T, Yoshie O. Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7. J Biol Chem. 1998; 273(12):7118-7122. (Biology). View Reference
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565869 Rev. 3

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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.