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Analysis of Stat3 (pY705) expressed in human peripheral blood lymphocytes. 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. Cells were lysed and 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 and then stained with BD Horizon™ R718 Mouse Anti-Stat3 (pY705) antibody (Cat. No. 567601). 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 LSRFortessa™ Cell Analyzer System and FlowJo™ software.
BD Phosflow™ R718 Mouse Anti-Stat3 (pY705)
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Preparation And Storage
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 CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- 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.
- 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).
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- The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity. However, verification testing has not been performed on all conjugate lots.
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Companion Products
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.
The antibody was conjugated to BD Horizon Red 718, which has been developed exclusively for BD Biosciences as a better alternative to Alexa Fluor® 700. BD Horizon Red 718 can be excited by the red laser (628 – 640 nm) and, with an Em Max around 718 nm, it can be detected using a 730/45 nm filter. Due to similar excitation and emission properties, we do not recommend using R718 in combination with APC-R700 or Alexa Fluor® 700.
Development References (5)
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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
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Imada K, Leonard WJ. The Jak-STAT pathway. Mol Immunol. 2000; 37:1-11. (Biology). View Reference
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Liu KD, Gaffen SL, Goldsmith MA. JAK/STAT signaling by cytokine receptors. Curr Opin Immunol. 1998; 10(3):271-278. (Biology). View Reference
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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
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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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For Research Use Only. Not for use in diagnostic or therapeutic procedures.