Description
The monoclonal antibody 4B12/CCR7 reacts with the mouse C-C chemokine receptor type 7 (CCR7). CCR7 is also known as CD197 (previously known as EBI1, Ebi1h and CMKBR7) and plays a central role in mediating homeostatic B and T lymphocyte trafficking to and within secondary lymphoid tissues. CD197 is a seven-transmembrane, G-protein-coupled, 43 kDa glycoprotein receptor that is specific for the CC chemokines, MIP3ß/Exodus-3/ELC/CKb11/Scya19/CCL19 and 6Ckine/Exodus-2/SLC/TCA4/CKb9/Scya21/CCL21. The mouse Ccr7 gene is located on chromosome 11. CD197 (CCR7) is differentially expressed by subsets of thymocytes. Positive CD197 expression appears to be involved in the cortex-to-medulla migration of positively-selected thymocytes wherein they complete functional maturation including the establishment of central tolerance. It is most highly expressed by some mature medullary single-positive thymocytes. CD197 is also expressed by subsets of mature peripheral CD4+ and CD8+ T lymphocytes including naïve and regulatory T cells and central memory T cells. In addition, it is differentially expressed by subsets of B lymphocytes, dendritic cells, and Langerhans cells. CD197 serves as a homing receptor that helps guide these various cell types to and within lymphoid tissues. In this way, CCR7 supports protective immunity while safeguarding self tolerance. Reportedly, the 4B12/CCR7 antibody is not agonistic, is not blocked by CCL21 nor by physiologic levels of CCL19, nor does the antibody block the binding of CCL21 to CCR7. The immunogen used to generate the 4B12 hybridoma was a mouse CCR7-transfected rat cell line.
Bead-based compensation or unmixing controls, such as BD® CompBeads or BD™ SpectraComp™, can be used as surrogates to assess fluorescence spillover when bound to fluorochrome-conjugated antibodies. Although these beads have spectral properties similar to cells, variations in spectral emission may occur, resulting in differing spillover values compared to biological controls. Therefore, it is considered best practice to compare the spillover obtained from cells and bead-based controls when using BD® CompBeads or BD™ SpectraComp™ for the first time, to ensure they are appropriate for the intended application.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions that minimize unconjugated dye and antibody. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. 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.
3. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
5. An isotype control should be used at the same concentration as the antibody of interest.
6. The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
8. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
9. For U.S. patents that may apply, see bd.com/patents.
10. When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
11. Please observe the following precautions: We recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to protect exposure of conjugated reagents, including cells stained with those reagents, to any room illumination. Absorption of visible light can significantly affect the emission spectra and quantum yield of tandem fluorochrome conjugates.
12. Tandem fluorochromes contain both an energy donor and an energy acceptor. 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 the donor may be observed. Additionally, multi-laser cytometers may directly excite both the donor and acceptor fluorochromes. Therefore, we recommend for every tandem conjugate, a matched individual single-stain control be acquired for generating a compensation or spectral unmixing matrix.
