The 5E6 (also known as clone SW5E6) antibody reacts with Ly-49C[BALB], Ly-49C[B6], Ly-49C[NZB], and Ly-49I[B6], inhibitory receptors which are expressed on subsets of natural killer (NK) cells and NK-1.1+ (or DX5+) T lymphocytes (NK-T cells) in all strains tested except C57BR and RIII, on a population of memory CD8+ T lymphocytes and NK1.1+ γδ T cells in C57BL/6 mice, and on a distinct subset of B-1 cells of BALB/c and C57BL/6 mice. The proportion of NK T cells expressing Ly-49C/I is higher (2-5 fold) in thymus than in liver (immature and mature NK T cells, respectively), and there is evidence that the down-regulation of Ly-49 receptor expression is necessary for normal NK T-cell development. Most NK cells express a single allele of Ly-49C, although occasionally they may express more than one allele. The Ly-49 family of NK-cell receptors are disulfide-linked type-II transmembrane protein homodimers with extracellular carbohydrate-recognition domains (CRD) that bind to MHC class I alloantigens. The Ly-49 family members are expressed independently, such that an individual NK or T cell may display more than one class of Ly-49 receptor homodimers. The 5E6 antibody is specific for the Ly-49C CRD. The Ly-49C[BALB] and Ly-49C[B6] alloantigens bind to MHC class I antigens of the b, d, k, and s haplotypes, and the 5E6 antibody blocks this binding. Binding of Ly-49C[BALB]- and Ly-49C[B6]- expressing transfectants to lymphoblasts of H-2[f], H-2[q], H-2[r], and H-2[v] strains has also been detected. Ly-49I[B6] transfectants bind H-2[r] lymphoblasts and bind much more weakly to the b, d, k, q, s, and v haplotypes. The levels of the Ly-49 inhibitory receptors are down-regulated by their ligands in vivo, and the various levels of expression of an Ly-49 inhibitory receptor may affect the specificity of NK cells. Ly-49C is specifically downregulated in the presence of H-2K[b] class I molecules (one of the Ly-49C ligands). Ly-49C[+] and/or Ly-49I[+] cells mediate allogeneic and hybrid resistance to H-2d bone marrow transplantation. In vitro and in vivo studies suggest that the Ly-49C and/or Ly-49I receptors mediate negative regulation of NK-cell cytolytic activity via tyrosine phosphorylation of their ITIMs (Immunoreceptor Tyrosine-based Inhibitory Motifs). The epitope recognized by this antibody on Ly49C may be masked on freshly isolated primary NK cells due to cis interactions with MHC class I molecules. This observation has been reported for other Ly49C monoclonal antibodies that bind to the same structural region.
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.
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 to the dye under optimum conditions that minimize unconjugated dye and antibody.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
3. For U.S. patents that may apply, see bd.com/patents.
4. 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.
5. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
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. 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.
8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
9. An isotype control should be used at the same concentration as the antibody of interest.
10. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
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.
