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      RY586 Rat Anti-Mouse TNF
      RY586 Rat Anti-Mouse TNF
      Two color flow cytometric analysis of TNF expression by stimulated Mouse splenocytes. Mouse splenic leucocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with stain buffer, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). After washing the cells were permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723) then stained in this buffer with APC Rat Anti-Mouse CD4 antibody (Cat. No. 561091/553051) and with either BD Horizon™ RY586 Rat IgG1, κ Isotype Control (Cat. No. 568822; Left Plot) or BD Horizon RY586 Rat Anti-Mouse TNF antibody (Cat. No. 568547/568548; Right Plot) at 0.25 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
      RY586 Rat Anti-Mouse TNF
      Two color flow cytometric analysis of TNF expression by stimulated Mouse splenocytes. Mouse splenic leucocytes were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (PMA; Sigma P-8139; 50 ng/ml) and Ionomycin (Sigma I-0634; 1 μg/ml) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with stain buffer, and fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655). After washing the cells were permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723) then stained in this buffer with APC Rat Anti-Mouse CD4 antibody (Cat. No. 561091/553051) and with either BD Horizon™ RY586 Rat IgG1, κ Isotype Control (Cat. No. 568822; Left Plot) or BD Horizon RY586 Rat Anti-Mouse TNF antibody (Cat. No. 568547/568548; Right Plot) at 0.25 µg/test using BD Biosciences Intracellular Cytokine Staining protocol. The bivariate pseudocolor density plot showing the correlated expression of TNF (or Ig Isotype control staining) versus CD4 was derived from gated events with the forward and side light-scatter characteristics of intact stimulated leucocytes. Flow cytometric analysis was performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software. Data shown on this Technical Data Sheet are not lot specific.
      Product Details
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      BD Horizon™
      Tnf; Tnfa; TNF alpha; TNF-a; Tnfsf1a; Tnfsf2; TNFSF2; Cachectin; DIF
      Mouse (QC Testing)
      Rat IgG1
      Recombinant Mouse TNF
      Intracellular staining (flow cytometry) (Routinely Tested)
      0.2 mg/ml
      21926
      Aqueous buffered solution containing ≤0.09% sodium azide.
      RUO


      Preparation And Storage

      The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed. Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
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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 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.

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      Product Notices

      1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      2. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
      3. An isotype control should be used at the same concentration as the antibody of interest.
      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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      6. CF™ is a trademark of Biotium, Inc.
      7. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
      8. For U.S. patents that may apply, see bd.com/patents.
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      568547 Rev. 2
      Antibody Details
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      MP6-XT22

      The MP6-XT22 antibody specifically binds to mouse Tumor Necrosis Factor (TNF, also known as TNF-α).  TNF is produced by many activated cell types including monocytes, macrophages, astrocytes, granulocytes, mast cells, T and B lymphocytes, NK cells, keratinocytes, fibroblasts, adipocytes, and certain tumor cells. Activated cells express type II transmembrane TNF glycoproteins that associate as homotrimeric complexes. After enzymatic cleavage, the extracellular regions of membrane TNF are shed as soluble homotrimers. TNF is a potent multifunctional cytokine that can exert regulatory and cytotoxic effects on a wide range of normal lymphoid and non-lymphoid cells and tumor cells. Although TNF serves as a primary mediator in protective immune responses against microbial and viral pathogens, it can also drive systemic pathophysiologic responses including septic shock, cachexia and autoimmune diseases. Mouse TNF exerts its biological activities by binding and signaling through cell surface membrane Type I and Type II TNF Receptors (aka, TNFRI/CD120a and TNFRII/CD120b, respectively).

      568547 Rev. 2
      Format Details
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      RY586
      The BD Horizon RealYellow™ 586 (RY586) Dye is part of the BD family of yellow-green dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 565-nm and an emission maximum (Em Max) at 586-nm. Driven by BD innovation, RY586 can be used on both spectral and conventional cytometers and is designed to be excited by the Yellow-Green laser (561-nm) with minimal excitation by the 488-nm Blue laser. For conventional instruments equipped with a Yellow-Green laser (561-nm), RY586 can be used as an alternative to PE and we recommend using an optical filter centered near 586-nm (eg, a 586/15-nm bandpass filter). For spectral instruments equipped with a Yellow-Green laser (561-nm), it can be used in conjunction with PE. Compared to PE, RY586 is similar in brightness, minimal spillover into Blue detectors, and increased spillover into the 610/20-nm (PE-CF594) detector. Please ensure that your instrument configuration (lasers and optical filters) is appropriate for this dye.
      altImg
      RY586
      Yellow-Green 561 nm
      564 nm
      586 nm
      568547 Rev.2
      Citations & References
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      Development References (7)

      1. Abrams J. Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies. Curr Protoc Immunol. 2001; 1:6.20-6.21. (Clone-specific: ELISA). View Reference
      2. Abrams JS, Roncarolo MG, Yssel H, Andersson U, Gleich GJ, Silver JE. Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples. Immunol Rev. 1992; 127:5-24. (Clone-specific: Blocking, ELISA). View Reference
      3. Bitsaktsis C, Winslow G. Fatal recall responses mediated by CD8 T cells during intracellular bacterial challenge infection. J Immunol. 2006; 177(7):4644-4651. (Clone-specific: Blocking). View Reference
      4. Hunter CA, Litton MJ, Remington JS, Abrams JS. Immunocytochemical detection of cytokines in the lymph nodes and brains of mice resistant or susceptible to toxoplasmic encephalitis. J Infect Dis. 1994; 170(4):939-945. (Clone-specific: Immunohistochemistry). View Reference
      5. Litton MJ, Sander B, Murphy E, O'Garra A, Abrams JS. Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B. J Immunol Methods. 1994; 175(1):47-58. (Clone-specific: Immunohistochemistry). View Reference
      6. Prussin C, Metcalfe DD. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies. J Immunol Methods. 1995; 188(1):117-128. (Methodology: Flow cytometry, IC/FCM Block). View Reference
      7. Yang J, Kawamura I, Zhu H, Mitsuyama M. Involvement of natural killer cells in nitric oxide production by spleen cells after stimulation with Mycobacterium bovis BCG. Study of the mechanism of the different abilities of viable and killed BCG. J Immunol. 1995; 155(12):5728-5735. (Clone-specific: Blocking). View Reference
      View All (7) View Less
      568547 Rev. 2

      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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