Description
The 5H10-1 monoclonal antibody specifically recognizes CD8a which is also known as CD8 alpha (CD8α), Ly-2 or Lyt-2. CD8a is an ~34 kDa single pass type I transmembrane glycoprotein that is encoded by Cd8a (CD8 antigen, alpha chain) which belongs to the immunoglobulin superfamily (IgSF). CD8a is comprised of an extracellular region that contains an IgV-like domain followed by a transmembrane region and cytoplasmic tail with a Lck tyrosine kinase binding motif. CD8a is expressed on the cell surface as either a disulfide bond-linked homodimer (CD8αα) or a heterodimer (CD8αβ) when disulfide bonded to CD8b (also known as, CD8 beta/CDβ, Ly-3, or Lyt-3), another type I transmembrane glycoprotein. CD8aβ is expressed on most thymocytes and a subpopulation of mature peripheral TCR αβ T cells including some intraepithelial lymphocytes (IELs). CD8αα is expressed on IELs which include either TCR αβ or TCR γδ T cells as well as on other leucocytes, such as, subsets of NK cells and dendritic cells (DCs). CD8αβ serves as a co-receptor for antigen-stimulated CD8+ T cells by binding to the same peptide:MHC class I complex as the T cell receptor and by helping to trigger the subsequent signaling cascade through Lck activation upon antigen recognition. Through high-affinity binding to nonclassical MHC class-Ib molecules, CD8αα can reportedly function less efficiently as a co-receptor and even repress antigen-stimulated CD8+ T cell responses.
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 cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular 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.
