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      RB705 Mouse Anti-Stat3 (pY705)
      RB705 Mouse Anti-Stat3 (pY705)
      Analysis of Stat3 (pY705) expressed in Human peripheral blood lymphocytes.  Human whole blood was either left unstimulated (dashed line histogram) or stimulated (solid line histogram) with 100 ng/ml BD Pharmingen™ Recombinant Human IL-6 protein (Cat. No. 550071) for 15 minutes at 37°C. Erythrocytes were lysed and the leukocytes were fixed in a single step using BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 min at 37 °C. Cells were then permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, washed twice with 5-10 mL of BD Pharmingen™ Stain Buffer (FBS) and then stained with BD Phosflow™ RB705 Mouse Anti-Stat3 (pY705) antibody (Cat. No. 570575/570663). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software.
      RB705 Mouse Anti-Stat3 (pY705)
      Analysis of Stat3 (pY705) expressed in Human peripheral blood lymphocytes.  Human whole blood was either left unstimulated (dashed line histogram) or stimulated (solid line histogram) with 100 ng/ml BD Pharmingen™ Recombinant Human IL-6 protein (Cat. No. 550071) for 15 minutes at 37°C. Erythrocytes were lysed and the leukocytes were fixed in a single step using BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 min at 37 °C. Cells were then permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, washed twice with 5-10 mL of BD Pharmingen™ Stain Buffer (FBS) and then stained with BD Phosflow™ RB705 Mouse Anti-Stat3 (pY705) antibody (Cat. No. 570575/570663). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD FACSymphony™ A5 SE Cell Analyzer System and FlowJo™ Software.
      Product Details
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      BD Phosflow™
      Acute-phase response factor, APRF
      Human (QC Testing), Mouse (Tested in Development)
      Mouse IgG2a, κ
      Phosphorylated Human Stat3 Peptide
      Intracellular staining (flow cytometry) (Routinely Tested)
      5 µl/test
      6774, 20848
      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. 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.
      2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
      3. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
      4. An isotype control should be used at the same concentration as the antibody of interest.
      5. 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.
      6. 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.
      7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
      8. 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.
      9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
      10. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
      11. Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
      12. For U.S. patents that may apply, see bd.com/patents.
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      570663 Rev. 1
      Antibody Details
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      4/P-STAT3

      Stat (Signal transducer and activators of transcription) proteins are critical mediators of the biologic activity of cytokines, including interleukins, interferons, erythropoietin, and growth factors.  Ligand-receptor interaction leads to activation of constitutively associated JAK family kinases and subsequent recruitment/activation of Stat proteins by tyrosine phosphorylation.  Active Stat proteins then move to the nucleus to promote transcription of cytokine-inducible genes.  Seven Stat proteins have been cloned, each of which is differentially expressed and/or activated in a cytokine-specific and cell type-specific manner. Stat3 is a 92-kDa protein that is activated as a DNA- binding protein through cytokines, such as IL-6, and growth factors, such as EGF. Stat3 activation occurs via tyrosine phosphorylation at Y705. Tyrosine phosphorylation in response to cytokine stimulation is generally mediated by JAK1. Upon activation, Stat3 dimerizes, translocates to the nucleus and binds DNA response elements, thereby regulating gene expression. It has been reported that Stat3 binds to DNA as a homodimer, but it is also capable of binding as a heterodimer with Stat1. In addition to tyrosine phosphorylation, Stat3 is also phosphorylated at S727 via the MAPK pathway.  Stat3 is widely expressed and can bind to the sis-inducible element (SIE) site from the c-fos promoter. This site is similar to the GAS element that is present in IFN-γ induced genes. Thus, phosphorylation of Y705 in Stat3 occurs in response to growth factors and cytokines, and is essential for normal transcription activity.

      The 4/P-STAT3 monoclonal antibody recognizes the phosphorylated Y705 of Stat3.

      570663 Rev. 1
      Format Details
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      RB705
      The BD Horizon RealBlue™ 705 (RB705) Dye is part of the BD® family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 707-nm as measured using an antibody-dye conjugate. Driven by BD® innovation, RB705 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB705 can be used as an alternative to PerCP-Cy5.5 or BB700 and we recommend using an optical filter centered near 710-nm (e.g., a 695/40 or 710/50-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PerCP-Cy5.5. RB705 is on average brighter than PerCP-Cy5.5 and BB700, and has minimal spillover into Yellow-Green detectors.
      altImg
      RB705
      Blue 488 nm
      498 nm
      707 nm
      570663 Rev.1
      Citations & References
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      View product citations for antibody "570663" on CiteAb

      Development References (5)

      1. Bromberg J, Darnell JE. The role of STATs in transcriptional control and their impact on cellular function. Oncogene. 2000; 19(21):2468-2473. (Biology). View Reference
      2. Imada K, Leonard WJ. The Jak-STAT pathway. Mol Immunol. 2000; 37:1-11. (Biology). View Reference
      3. Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
      4. Renner ED, Rylaarsdam S, Anover-Sombke S, et al. Novel signal transducer and activator of transcription 3 (STAT3) mutations, reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in hyper-IgE syndrome. J Allergy Clin Immunol. 2008; 122(1):181-187. (Clone-specific: Flow cytometry). View Reference
      5. Tanaka S, Saito Y, Kunisawa J, et al. Development of mature and functional human myeloid subsets in hematopoietic stem cell-engrafted NOD/SCID/IL2rgammaKO mice. J Immunol. 2012; 188(12):6145-6155. (Clone-specific: Flow cytometry). View Reference
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      570663 Rev. 1

       

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      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.