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APC-Cy™7 Rat Anti-Mouse Ly-6A/E
APC-Cy™7 Rat Anti-Mouse Ly-6A/E

Flow cytometric analysis of Ly-6A/E on stimulated mouse splenocytes.  BALB/c splenocytes were stimulated with 2.5-5.0 µg/mL concanavilin A (Sigma-Aldrich cat. no. C2010) for 48 hours and were subsequently stained either with a APC-Cy™7 Rat IgG2a, κ isotype control (Cat. No. 552770, unshaded) or with the APC-Cy™7 Rat Anti-Mouse Ly-6A/E antibody (Cat. No. 560654, shaded).  Histograms were derived from gated events based on light scattering characteristics for splenocytes.  Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Flow cytometric analysis of Ly-6A/E on stimulated mouse splenocytes.  BALB/c splenocytes were stimulated with 2.5-5.0 µg/mL concanavilin A (Sigma-Aldrich cat. no. C2010) for 48 hours and were subsequently stained either with a APC-Cy™7 Rat IgG2a, κ isotype control (Cat. No. 552770, unshaded) or with the APC-Cy™7 Rat Anti-Mouse Ly-6A/E antibody (Cat. No. 560654, shaded).  Histograms were derived from gated events based on light scattering characteristics for splenocytes.  Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Product Details
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BD Pharmingen™
Ly-6A/E; Lymphocyte antigen 6A-2/6E-1; Ly-6A.2/Ly-6E.1; Sca-1; TAP
Mouse (QC Testing)
Rat LEW, also known as Lewis IgG2a, κ
IL-2-dependent mouse T-cell line CTL-L
Flow cytometry (Routinely Tested)
0.2 mg/ml
110454
AB_1727552
Aqueous buffered solution containing protein stabilizer and ≤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 APC-Cy7 under optimum conditions, and unconjugated antibody and free APC-Cy7 were removed.

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. Warning: Some APC-Cy7 and PE-Cy7 conjugates show changes in their emission spectrum with prolonged exposure to formaldehyde. If you are unable to analyze fixed samples within four hours, we recommend that you use BD™ Stabilizing Fixative (Cat. No. 338036).
  5. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  6. APC-Cy7 tandem fluorochrome emission is collected in a detector for fluorescence wavelengths of 750 nm and higher.
  7. APC-Cy7 is a tandem fluorochrome composed of Allophycocyanin (APC), which is excited by laser lines between 595 and 647 nm and serves as an energy donor, coupled to the cyanine dye Cy7™, which acts as an energy acceptor and fluoresces at 780 nm. BD Biosciences Pharmingen has maximized the fluorochrome energy transfer in APC-Cy7, thus maximizing its fluorescence emission intensity, minimizing residual emission from APC, and minimizing required electronic compensation in multilaser-laser flow cytometry systems. Note: Although every effort is made to minimize the lot-to-lot variation in residual emission from APC, it is strongly recommended that every lot be tested for differences in the amount of compensation required and that individual compensation controls are run for each APC-Cy7 conjugate.
  8. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  9. Cy is a trademark of GE Healthcare.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560654 Rev. 2
Antibody Details
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D7

The D7 monoclonal antibody recognizes Ly-6A.2 and Ly-6E.1, which are allelic members of the Ly-6 multigene family. Sca-1 (Ly6A/E), a phosphatidylinositol-anchored protein of about 18 kDa, is expressed on the multipotent hematopoietic stem cells (HSC) in the bone marrow of mice with both Ly-6 haplotypes. In mice expressing the Ly-6.2 haplotype (e.g., AKR, C57BL, C57BR, C57L, C58, DBA/2, PL, SJL, SWR, 129), Ly-6A/E is also expressed on distinct subpopulations of bone marrow and peripheral B lymphocytes as well as thymic and peripheral T lymphocytes. Strains with the Ly-6.1 haplotype (e.g., A, BALB/c, CBA, C3H/He, DBA/1, NZB) have few Ly-6A/E+ resting peripheral lymphocytes; activation of lymphocytes from mice of both Ly-6 haplotypes leads to strong expression of the Sca-1 antigen. Studies with the D7 antibody have demonstrated that Ly-6A/E may be involved in the regulation of B and T lymphocyte responses, and appears to be required for T-cell receptor-mediated T-cell activation. The purified E13-161.7 mAb (anti-Ly-6A/E) can block binding of FITC-conjugated D7 antibody to mouse splenocytes, but purified mAb D7 is unable to block binding of FITC-conjugated E13-161.7 antibody. Anti-Ly-6A/E (Sca-1) mAb may be used in combination with a Mouse Lineage Panel of antibodies to identify HSC.

560654 Rev. 2
Format Details
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APC-Cy7
APC-Cy7 dye is a part of the BD APC red family of dyes. This tandem fluorochrome is comprised of a Allophycocyanin (APC) donor that has excitation maxima (Ex Max) of 651 nm and an acceptor dye, Cy™7, with an emission maximum (Em Max) at 779 nm. APC-Cy7 can be excited by the Red (627-640 nm) laser and detected using an optical filter centered near 780 nm (e.g., a 760/60 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
APC-Cy7
Red 627-640 nm
651 nm
779 nm
560654 Rev.2
Citations & References
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Development References (25)

  1. Auerbach R, Huang H, Lu L. Hematopoietic stem cells in the mouse embryonic yolk sac. Stem Cells. 1996; 14(3):269-280. (Biology). View Reference
  2. Codias EK, Cray C, Baler RD, Levy RB, Malek TR. Expression of Ly-6A/E alloantigens in thymocyte and T-lymphocyte subsets: variability related to the Ly-6a and Ly-6b haplotypes. Immunogenetics. 1989; 29(2):98-107. (Biology). View Reference
  3. Codias EK, Malek TR. Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules. J Immunol. 1990; 144(6):2197-2204. (Biology: Activation). View Reference
  4. Codias EK, Rutter JE, Fleming TJ, Malek TR. Down-regulation of IL-2 production by activation of T cells through Ly-6A/E. J Immunol. 1990; 145(5):1407-1414. (Biology: Activation). View Reference
  5. Fleming TJ, Malek TR. Multiple glycosylphosphatidylinositol-anchored Ly-6 molecules and transmembrane Ly-6E mediate inhibition of IL-2 production. J Immunol. 1994; 153(5):1955-1962. (Biology). View Reference
  6. Flood PM, Dougherty JP, Ron Y. Inhibition of Ly-6A antigen expression prevents T cell activation. J Exp Med. 1990; 172(1):115-120. (Biology). View Reference
  7. Ito M, Anan K, Misawa M, Kai S, Hara H. In vitro differentiation of murine Sca-1+Lin- cells into myeloid, B cell and T cell lineages. Stem Cells. 1996; 14(4):412-418. (Biology). View Reference
  8. Ivanov V, Fleming TJ, Malek TR. Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E. J Immunol. 1994; 153(6):2394-2406. (Biology). View Reference
  9. Jurecic R, Van NT, Belmont JW. Enrichment and functional characterization of Sca-1+WGA+, Lin-WGA+, Lin-Sca-1+, and Lin-Sca-1+WGA+ bone marrow cells from mice with an Ly-6a haplotype. Blood. 1993; 82(9):2673-2683. (Biology). View Reference
  10. Malek TR, Danis KM, Codias EK. Tumor necrosis factor synergistically acts with IFN-gamma to regulate Ly-6A/E expression in T lymphocytes, thymocytes and bone marrow cells. J Immunol. 1989; 142(6):1929-1936. (Biology). View Reference
  11. Malek TR, Ortega G, Chan C, Kroczek RA, Shevach EM. Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies. J Exp Med. 1986; 164(3):709-722. (Biology). View Reference
  12. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A. 1995; 92(22):10302-10306. (Biology). View Reference
  13. Morrison SJ, Wandycz AM, Hemmati HD, Wright DE, Weissman IL. Identification of a lineage of multipotent hematopoietic progenitors. Development. 1997; 124(10):1929-1939. (Biology). View Reference
  14. Morrison SJ, Wright DE, Weissman IL. Cyclophosphamide/granulocyte colony-stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc Natl Acad Sci U S A. 1997; 94(5):1908-1913. (Biology). View Reference
  15. Ortega G, Korty PE, Shevach EM, Malek TR. Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity. J Immunol. 1986; 137(10):3240-3246. (Immunogen: Flow cytometry, Immunoprecipitation, Western blot). View Reference
  16. Osawa M, Nakamura K, Nishi N, et al. In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/-) hemopoietic stem cells. J Immunol. 1996; 156(9):3207-3214. (Biology). View Reference
  17. Palfree RG, Dumont FJ, Hammerling U. Ly-6A.2 and Ly-6E.1 molecules are antithetical and identical to MALA-1. Immunogenetics. 1986; 23(3):197-207. (Biology). View Reference
  18. Rock KL, Reiser H, Bamezai A, McGrew J, Benacerraf B. The LY-6 locus: a multigene family encoding phosphatidylinositol-anchored membrane proteins concerned with T-cell activation. Immunol Rev. 1989; 111:195-224. (Biology: Flow cytometry). View Reference
  19. Spangrude GJ, Aihara Y, Weissman IL, Klein J. The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets. J Immunol. 1988; 141(11):3697-3707. (Biology). View Reference
  20. Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood. 1993; 82(11):3327-3332. (Biology). View Reference
  21. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science. 1988; 241(4861):58-62. (Biology). View Reference
  22. Spangrude GJ, Klein J, Heimfeld S, Aihara Y, Weissman IL. Two monoclonal antibodies identify thymic-repopulating cells in mouse bone marrow. J Immunol. 1989; 142(2):425-430. (Biology). View Reference
  23. Yamamoto Y, Yasumizu R, Amou Y, et al. Characterization of peripheral blood stem cells in mice. Blood. 1996; 88(2):445-454. (Biology). View Reference
  24. Yonemura Y, Ku H, Lyman SD, Ogawa M. In vitro expansion of hematopoietic progenitors and maintenance of stem cells: comparison between FLT3/FLK-2 ligand and KIT ligand. Blood. 1997; 89(6):1915-1921. (Biology). View Reference
  25. van de Rijn M, Heimfeld S, Spangrude GJ, Weissman IL. Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci U S A. 1989; 86(12):4634-4638. (Biology). View Reference
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560654 Rev. 2

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