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      BB515 Rat Anti-Mouse CD44
      BB515 Rat Anti-Mouse CD44
      Flow cytometric analysis of CD44 expression on mouse bone-marrow cells - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. Mouse bone-marrow cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with either BB515 Rat IgG2b, κ Isotype Control (Cat. No. 564421; dashed line histogram) or BD Horizon BB515 Rat Anti-Mouse CD44 antibody (Cat. No. 564587; bold solid line histogram). Alternatively, cells were stained with FITC Rat Anti-Mouse CD44 antibody (Cat. No. 553133/561859; thin solid line histogram). Overlaid histograms are shown to facilitate staining comparisons between: BB515 Anti-CD44 antibody versus its Ig Isotype Control (Left Panel), and BB515 Anti-CD44 antibody versus FITC Anti-CD44 antibody (Right Panel). The fluorescence histograms showing CD44 expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of viable bone marrow cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
      BB515 Rat Anti-Mouse CD44
      Flow cytometric analysis of CD44 expression on mouse bone-marrow cells - Staining comparisons between BD Horizon™ BB515- and FITC-conjugated antibodies. Mouse bone-marrow cells were preincubated with Purified Rat Anti-Mouse CD16/CD32 antibody (Mouse BD Fc Block™) (Cat. No. 553141/553142). The cells were then stained with either BB515 Rat IgG2b, κ Isotype Control (Cat. No. 564421; dashed line histogram) or BD Horizon BB515 Rat Anti-Mouse CD44 antibody (Cat. No. 564587; bold solid line histogram). Alternatively, cells were stained with FITC Rat Anti-Mouse CD44 antibody (Cat. No. 553133/561859; thin solid line histogram). Overlaid histograms are shown to facilitate staining comparisons between: BB515 Anti-CD44 antibody versus its Ig Isotype Control (Left Panel), and BB515 Anti-CD44 antibody versus FITC Anti-CD44 antibody (Right Panel). The fluorescence histograms showing CD44 expression (or Ig Isotype control staining) were derived from gated events with the forward and side light-scatter characteristics of viable bone marrow cells. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.
      Product Details
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      BD Horizon™
      Pgp-1; Ly-24; H-CAM; HERMES; ECMR-III; Hyaluronate Receptor
      Mouse (QC Testing)
      Rat IgG2b, κ
      Dexamethasone-induced, SJL mouse spontaneous myeloid leukemia M1 cells myeloid leukemia M1
      Flow cytometry (Routinely Tested)
      0.2 mg/ml
      12505
      AB_2738855
      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 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).

              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.

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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. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
      6. 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.
      7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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      564587 Rev. 3
      Antibody Details
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      IM7

                The IM7 antibody specifically recognizes an epitope on both alloantigens and all isoforms of the CD44 glycoprotein (Pgp-1, Ly-24). The standard form of CD44, lacking variable exons and referred to as CD44H or CD44s, is widely expressed on hematopoietic and non-hematopoietic cells. CD44 isoforms encoded by variable exons are expressed on epithelial cells, but only at low levels on most leukocytes. Mice with the Ly-24.1 alloantigen (e.g., BALB/c, CBA/J, DBA/1, DBA/2) have relatively large subsets of CD44H+ T lymphocytes, while Ly-24.2 strains (e.g., A, AKR, CBA/N, C3H/He, C57BL, C57BR, C57L, C58, NZB, SJL, SWR, 129) have fewer CD44H+ T cells. CD44 is a cell adhesion receptor, and its principal ligand, hyaluronate, is a common component of extracellular matrices. Differential glycosylation of CD44 influences its binding to hyaluronate.  Additional ligands include the cell surface form of CD74 and the cytokine osteopontin (Eta-1). Bone marrow- and thymus-derived progenitor cells capable of repopulating the thymus express CD44. In the periphery, the level of CD44 expression increases upon activation of B lymphocytes, CD4+ T cells, and CD8+ T cells; memory cells can be recognized by their CD44[hi] phenotype. The IM7 mAb inhibits established collagen-induced arthritis in DBA/1 mice. Moreover, it prevents CNS inflammation and clinical symptoms of experimental autoimmune encephalomyelitis. In contrast, the same antibody exacerbates experimental autoimmune thyroiditis in CBA/J mice. The IM7 mAb recognizes a different epitope from that recognized by mAb KM114, and the antibody pair can be used in ELISA to detect soluble CD44. It has been observed that IM7 antibody crossreacts with human, dog, cat, horse, cow, and pig leukocytes. Anti-human CD44, clone G44-26, and IM7 antibody compete for binding to human peripheral blood lymphocytes.

              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.

      564587 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.
      altImg
      BB515
      Blue 488 nm
      490 nm
      515 nm
      564587 Rev.3
      Citations & References
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      Development References (15)

      1. Brocke S, Piercy C, Steinman L, Weissman IL, Veromaa T. Antibodies to CD44 and integrin alpha4, but not L-selectin, prevent central nervous system inflammation and experimental encephalomyelitis by blocking secondary leukocyte recruitment. Proc Natl Acad Sci U S A. 1999; 96(12):6896-6901. (Clone-specific: Blocking). View Reference
      2. Budd RC, Cerottini JC, Horvath C, et al. Distinction of virgin and memory T lymphocytes. Stable acquisition of the Pgp-1 glycoprotein concomitant with antigenic stimulation. J Immunol. 1987; 138(10):3120-3129. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting, Immunoprecipitation). View Reference
      3. Camp RL, Scheynius A, Johansson C, Pure E. CD44 is necessary for optimal contact allergic responses but is not required for normal leukocyte extravasation. J Exp Med. 1993; 178(2):497-507. (Clone-specific: Induction, Inhibition, Radioimmunoassay). View Reference
      4. Ernst DN, Weigle WO, Noonan DJ, McQuitty DN, Hobbs MV. The age-associated increase in IFN-γ synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression. J Immunol. 1993; 151(2):575-587. (Clone-specific: Flow cytometry). View Reference
      5. Godfrey DI, Kennedy J, Suda T, Zlotnik A. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J Immunol. 1993; 150(10):4244-4252. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
      6. Hathcock KS, Hirano H, Murakami S, Hodes RJ. CD44 expression on activated B cells. Differential capacity for CD44-dependent binding to hyaluronic acid. J Immunol. 1993; 151(12):6712-6722. (Clone-specific: Flow cytometry, Immunoprecipitation). View Reference
      7. Hyman R, Lesley J, Schulte R, Trotter J. Progenitor cells in the thymus: most thymus-homing progenitor cells in the adult mouse thymus bear Pgp-1 glycoprotein but not interleukin-2 receptor on their cell surface. Cell Immunol. 1986; 101(2):320-327. (Clone-specific: Flow cytometry). View Reference
      8. Katoh S, McCarthy JB, Kincade PW. Characterization of soluble CD44 in the circulation of mice. Levels are affected by immune activity and tumor growth. J Immunol. 1994; 153(8):3440-3449. (Clone-specific: ELISA). View Reference
      9. Katoh S, Zheng Z, Oritani K, Shimozato T, Kincade PW. Glycosylation of CD44 negatively regulates its recognition of hyaluronan. J Exp Med. 1995; 182(2):419-429. (Clone-specific: Blocking). View Reference
      10. Lesley J, Hyman R, Kincade PW. CD44 and its interaction with extracellular matrix. Adv Immunol. 1993; 54:271-335. (Biology). View Reference
      11. Lesley J, Trowbridge IS. Genetic characterization of a polymorphic murine cell-surface glycoprotein. Immunogenetics. 1982; 15(3):313-320. (Clone-specific: Immunoaffinity chromatography, Immunoprecipitation, Radioimmunoassay). View Reference
      12. Lynch F, Ceredig R. Mouse strain variation in Ly-24 (Pgp-1) expression by peripheral T cells and thymocytes: implications for T cell differentiation. Eur J Immunol. 1989; 19(2):223-229. (Clone-specific: Flow cytometry). View Reference
      13. MacDonald HR, Budd RC, Cerottini JC. Pgp-1 (Ly 24) as a marker of murine memory T lymphocytes. Curr Top Microbiol Immunol. 1990; 159:97-109. (Biology). View Reference
      14. Trowbridge IS, Lesley J, Schulte R, Hyman R, Trotter J. Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid tissues. Immunogenetics. 1982; 15:299-312. (Immunogen: Cytotoxicity, Depletion, Flow cytometry, Functional assay, Immunoprecipitation). View Reference
      15. 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
      View All (15) View Less
      564587 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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