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R718 Mouse Anti-Stat1 (pY701)
R718 Mouse Anti-Stat1 (pY701)

Flow cytometric analysis of Stat1 (pY701) expression in human CD4+ T lymphocytes. Untreated (dashed line histogram) or treated (solid line histogram) peripheral blood mononuclear cells (PBMC) from the BD Phosflow™ T Cell Kit Lyophilized Cells (Catalog No. 560760) were reconstituted in BD Pharmingen™ Stain Buffer (Cat. No. 554656) for 15 minutes at room temperature. These cells are fixed, permeabilized, and lyophilized PBMC that are either untreated or treated with stimulators [including phorbol 12-myristate 13-acetate (PMA), interferon-alpha (IFN-α), interleukin-2 (IL-2), IL-4, and IL-6]. The reconstituted cells were then washed and stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 564724) and BD Phosflow™ R718 Mouse Anti-Stat1 (pY701) antibody (Cat. No. 566983/566984). Histograms showing Stat1 (pY701) expression were derived from CD4-positive gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.

Flow cytometric analysis of Stat1 (pY701) expression in human CD4+ T lymphocytes. Untreated (dashed line histogram) or treated (solid line histogram) peripheral blood mononuclear cells (PBMC) from the BD Phosflow™ T Cell Kit Lyophilized Cells (Catalog No. 560760) were reconstituted in BD Pharmingen™ Stain Buffer (Cat. No. 554656) for 15 minutes at room temperature. These cells are fixed, permeabilized, and lyophilized PBMC that are either untreated or treated with stimulators [including phorbol 12-myristate 13-acetate (PMA), interferon-alpha (IFN-α), interleukin-2 (IL-2), IL-4, and IL-6]. The reconstituted cells were then washed and stained with BD Horizon™ BUV395 Mouse Anti-Human CD4 antibody (Cat. No. 564724) and BD Phosflow™ R718 Mouse Anti-Stat1 (pY701) antibody (Cat. No. 566983/566984). Histograms showing Stat1 (pY701) expression were derived from CD4-positive gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry and data analysis were performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.

Product Details
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BD Phosflow™
Signal Transducer and Activator of Transcription-1; STAT91; ISGF-3; CANDF7
Human (QC Testing), Mouse (Tested in Development), Rat (Predicted)
Mouse IgG2a
Phosphorylated Human Stat1 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2869988
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 to the dye under optimum conditions and unreacted dye was removed.

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.

Product Notices

  1. 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).
  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. Alexa Fluor® is a registered trademark of Life Technologies Corporation.
  5. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  6. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  8. This product is provided under an Agreement between BIOTIUM and BD Biosciences. This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature. No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel. This product is for research use only. Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027. Fax: (510) 265-1352. Email: btinfo@biotium.com.
  9. Alexa Fluor™ is a trademark of Life Technologies Corporation.
567646 Rev. 1
Antibody Details
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4a

Stat (Signal transducer and activators of transcription) proteins mediate the biological activity of cytokines, including interleukins, interferons, erythropoietin, and growth factors.  Ligand-receptor interaction activates constitutively-associated JAK family kinases as well as subsequent recruitment and 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.  Stat1 and Stat2 are components of the ISGF3 (Interferon-Stimulated Gene Factor 3) complex, the primary transcription activator induced by interferon binding to a specific cell-surface receptor.  Stat1 has two alternatively spliced isoforms, 91-kDa Stat1α and 84-kDa Stat1β; Stat1α has 38 additional C-terminal amino acids.  In response to the binding of IFNα, IFNγ, EGF, PDGF, or CSF-1 to their respective receptors, the Stat1 subunits become tyrosine-phosphorylated at Y701, and the complex translocates to the nucleus. This forms an active complex that includes the DNA-binding p48 subunit, and is responsible for modulating interferon-stimulated genes (ISGs) transciption.

The 4a monoclonal antibody recognizes the phosphorylated Y701 in Stat1α and Stat1β.

The antibody was conjugated to BD Horizon™ Red 718, which has been developed exclusively by 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.

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Format Details
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R718
The BD Horizon™ Red 718 (R718) Dye is part of the BD red family of dyes. It is a small organic fluorochrome with an excitation maximum (Ex Max) at 695-nm and an emission maximum (Em Max) at 718-nm. Driven by BD innovation, R718 is designed to be excited by the red laser (627–640-nm) and detected using an optical filter centered near 720-nm (e.g., a 720/40-nm bandpass filter). R718 is a brighter alternative to Alexa Fluor™ 700. R718 is also a bright small molecule alternative to APC-R700 with lower spread into the APC detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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R718
Red 627-640 nm
695 nm
718 nm
567646 Rev.1
Citations & References
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Development References (6)

  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. Darnell JE Jr. STATs and gene regulation. Science. 1997; 277(5332):1630-1635. (Biology). View Reference
  3. Fu XY, Zhang JJ. Transcription factor p91 interacts with the epidermal growth factor receptor and mediates activation of the c-fos gene promoter. Cell. 1993; 74(6):1135-1145. (Biology). View Reference
  4. Perez OD, Mitchell D, Campos R, Gao GJ, Li L, Nolan GP. Multiparameter analysis of intracellular phosphoepitopes in immunophenotyped cell populations by flow cytometry. Curr Protoc Cytom. 2005; 6.20.1-6.20.22. (Clone-specific: Flow cytometry). View Reference
  5. Suni MA, Maino VC. Flow cytometric analysis of cell signaling proteins. Methods Mol Biol. 2011; 717:155-169. (Clone-specific). View Reference
  6. 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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567646 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.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product 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.