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      BB515 Rat Anti-Mouse CD8a
      BB515 Rat Anti-Mouse CD8a
      Two-color flow cytometric analysis of CD8a expression on mouse splenocytes. Mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Hamster Anti-Mouse CD3e (Cat. No. 561826/553066) and either BD Horizon™ BB515 Rat IgG2a, κ Isotype Control (Cat. No. 564418; Left Panel) or BD Horizon BB515 Rat Anti-Mouse CD8a antibody (Cat. No. 564422/564459; Right Panel) antibodies. The two-color fluorescence contour plots showing the correlated expression patterns of CD8a (or Ig Isotype control staining) versus CD3e were derived from gated events with the forward and side light-scatter characteristic of viable splenic leucocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
      BB515 Rat Anti-Mouse CD8a
      Two-color flow cytometric analysis of CD8a expression on mouse splenocytes. Mouse splenic leucocytes were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with APC Hamster Anti-Mouse CD3e (Cat. No. 561826/553066) and either BD Horizon™ BB515 Rat IgG2a, κ Isotype Control (Cat. No. 564418; Left Panel) or BD Horizon BB515 Rat Anti-Mouse CD8a antibody (Cat. No. 564422/564459; Right Panel) antibodies. The two-color fluorescence contour plots showing the correlated expression patterns of CD8a (or Ig Isotype control staining) versus CD3e were derived from gated events with the forward and side light-scatter characteristic of viable splenic leucocytes. Flow cytometry was performed using a BD™ LSR II Flow Cytometer System.
      Product Details
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      BD Horizon™
      Cd8a; CD8 alpha chain; Ly-2; Lyt2; Lyt-2; Ly-35; Ly-B
      Mouse (QC Testing)
      Rat LOU, also known as Louvain, LOU/C, LOU/M IgG2a, κ
      Mouse Spleen Cells or Thymocyte Membranes
      Flow cytometry (Routinely Tested)
      0.2 mg/ml
      12525
      AB_2738801
      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.
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      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 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.

      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).

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      Product Notices

      1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      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. 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.
      6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      564422 Rev. 3
      Antibody Details
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      53-6.7

              The 53-6.7 monoclonal antibody specifically binds to the 38 kDa α and 34 kDa α' chains of the CD8 differentiation antigen (Ly-2 or Lyt-2) of all mouse strains tested. The CD8 α and α' chains (CD8a) form heterodimers with the CD8 β chain (CD8b, Ly-3, or Lyt-3) on the surface of most thymocytes. A subpopulation of mature T lymphocytes (i.e., MHC class I-restricted T cells, including most T suppressor/cytotoxic cells) expresses almost exclusively the CD8 αβ heterodimer. Subsets of γδ TCR-bearing T cells, intestinal intrapithelial lymphocytes, and dendritic cells express CD8a without CD8b. It has been suggested that the expression of the CD8a/CD8b heterodimer is restricted to T lymphocytes which matured in the thymus or in an extrathymic environment that had been influenced by thymus-initiated neuroendocrine signals. CD8 is an antigen coreceptor on the T-cell surface which interacts with MHC class I molecules on antigen-presenting cells or epithelial cells. It participates in T-cell activation through its association with the T-cell receptor complex and protein tyrosine kinase lck (p56 [lck]). The CD8 α and α' chains arise from alternatively spliced messengers of a single CD8a gene. The longer α form associates with p56 [lck] via a CXCP motif in its cytoplasmic domain, which it shares with CD4, but not with CD8b. The truncated α' chain is unable to associate with p56 [lck], and it may function to attenuate the CD8-mediated costimulatory signal during intrathymic T-cell maturation.  In vivo and in vitro treatment with 53-6.7 mAb has reportedly been effective at depleting CD8+ peripheral T lymphocytes. The 53-6.7 antibody has also been reported to cross-react with CD8 α- and α'-like polypeptides on subsets of thymic and peripheral lymphocytes in the Egyptian toad, Bufo regularis.

      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.

      564422 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.
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      BB515
      Blue 488 nm
      490 nm
      515 nm
      564422 Rev.3
      Citations & References
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      Development References (18)

      1. Bierer BE, Sleckman BP, Ratnofsky SE, Burakoff SJ. The biologic roles of CD2, CD4, and CD8 in T-cell activation. Annu Rev Immunol. 1989; 7:579-599. (Biology). View Reference
      2. Fujiura Y, Kawaguchi M, Kondo Y, et al. Development of CD8 alpha alpha+ intestinal intraepithelial T cells in beta 2-microglobulin- and/or TAP1-deficient mice. J Immunol. 1996; 156(8):2710-2715. (Clone-specific: Flow cytometry). View Reference
      3. Hathcock KS. T cell depletion by cytotoxic elimination. Curr Protoc Immunol. 1991; 1:3.4.1-3.4.3. (Clone-specific: Cell separation, Depletion, Flow cytometry). View Reference
      4. Kruisbeek AM, Shevach EM. Proliferative assays for T cell function. Curr Protoc Immunol. 2004; 3:3.12.1-3.12.14. (Clone-specific: Depletion, In vivo exacerbation). View Reference
      5. LeFrancois L. Extrathymic differentiation of intraepithelial lymphocytes: generation of a separate and unequal T-cell repertoire. Immunol Today. 1991; 12(12):436-438. (Biology). View Reference
      6. Ledbetter JA, Herzenberg LA. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev. 1979; 47:63-90. (Immunogen: Flow cytometry, Immunofluorescence, Immunoprecipitation). View Reference
      7. Ledbetter JA, Rouse RV, Micklem HS, Herzenberg LA. T cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens. Two-parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views. J Exp Med. 1980; 152(2):280-295. (Immunogen: Flow cytometry). View Reference
      8. Ledbetter JA, Seaman WE, Tsu TT, Herzenberg LA. Lyt-2 and lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds. Relationship of subunits to T cell cytolytic activity. J Exp Med. 1981; 153(6):1503-1516. (Clone-specific: Blocking, Flow cytometry, Immunoprecipitation, Inhibition). View Reference
      9. Leishman AJ, Naidenko OV, Attinger A, et al. T cell responses modulated through interaction between CD8alphaalpha and the nonclassical MHC class I molecule, TL. Science. 2001; 294(5548):1848-1849. (Clone-specific: Blocking, Flow cytometry). View Reference
      10. MacDonald HR, Schreyer M, Howe RC, Bron C. Selective expression of CD8 alpha (Ly-2) subunit on activated thymic gamma/delta cells. Eur J Immunol. 1990; 20(4):927-930. (Biology). View Reference
      11. Nakayama K, Nakayama K, Negishi I, et al. Requirement for CD8 beta chain in positive selection of CD8-lineage T cells. Science. 1994; 263(5150):1131-1133. (Biology). View Reference
      12. Sydora BC, Brossay L, Hagenbaugh A, Kronenberg M, Cheroutre H. TAP-independent selection of CD8+ intestinal intraepithelial lymphocytes. J Immunol. 1996; 156(11):4209-4216. (Clone-specific: Flow cytometry). View Reference
      13. Takahashi K, Nakata M, Tanaka T, et al. CD4 and CD8 regulate interleukin 2 responses of T cells. Proc Natl Acad Sci U S A. 1992; 89(12):5557-5561. (Clone-specific: Immunoprecipitation, Inhibition). View Reference
      14. Vremec D, Zorbas M, Scollay R, et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med. 1992; 176(1):47-58. (Clone-specific: Flow cytometry). View Reference
      15. Wang J, Klein JR. Thymus-neuroendocrine interactions in extrathymic T cell development. Science. 1994; 265(5180):1860-1862. (Biology). View Reference
      16. Wu L, Vremec D, Ardavin C, et al. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur J Immunol. 1995; 25(2):418-425. (Biology). View Reference
      17. Zamoyska R, Derham P, Gorman SD, et al. Inability of CD8 alpha' polypeptides to associate with p56lck correlates with impaired function in vitro and lack of expression in vivo. Nature. 1989; 342(6247):278-281. (Biology). View Reference
      18. van Ewijk W, van Soest PL, van den Engh GJ. Fluorescence analysis and anatomic distribution of mouse T lymphocyte subsets defined by monoclonal antibodies to the antigens Thy-1, Lyt-1, Lyt-2, and T-200. J Immunol. 1981; 127(6):2594-2604. (Clone-specific: Flow cytometry, Immunofluorescence, Immunohistochemistry). View Reference
      View All (18) View Less
      564422 Rev. 3

      Please refer to Support Documents for Quality Certificates


      Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


      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.

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