The A1 monoclonal antibody specifically binds to the Ly-49A[B6] alloantigen, an inhibitory receptor that is expressed on subsets of natural killer (NK) cells and NK-1.1-positive T lymphocytes (NKT cells) in C57BL/6, C57BL/10, and B10 congenic mice, 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 (CD5+B220[lo]) of C57BL/6 mice. The A1 antibody has also been reported to crossreact with Ly-49ANOD, Ly-49PNOD, Ly-49P129/J, and Ly-49V129/J alloantigens. The proportion of NKT cells expressing Ly-49A is higher (2-5 fold) in thymus than in liver (immature and mature NKT cells, respectively), and there is evidence that the down regulation of Ly-49 receptor expression is necessary for normal NKT cell development to occur. Most NK cells express a single allele of Ly-49A, although occasionally they may express more than one allele. The Ly-49 family of NK-cell receptors, members of the C-type lectin superfamily, are disulfide-linked type-II transmembrane protein homodimers with extracellular carbohydrate-recognition domains (CRD) that bind to MHC class I alloantigens. The A1 antibody is specific for the Ly-49A[B6] CRD. 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 Ly-49A[B6] allonantigen binds to H-2D[d], H-2D[k], and H-2D[p], and the A1 antibody blocks this binding. Binding of Ly-49A[B6] to lyphoblasts expressing MHC class I antigens of the f, q, r, s, and v haplotypes has also been demonstrated. The levels of the Ly-49 inhibitory receptors are down-regulated by their ligands in vivo, and various levels of expression of a Ly-49 inhibitory receptor may affect the specificity of NK cells. In vitro studies suggest that the Ly-49A receptor mediates negative regulation of NK-cell cytolytic activity via tyrosine phosphorylation of its ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif).
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. 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.
11. 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.
12. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
13. Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
