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R718 Mouse Anti-Stat4 (pY693)
R718 Mouse Anti-Stat4 (pY693)

Flow cytometric analysis of Stat4 (pY693) expression in IFN-α treated human peripheral blood lymphocytes. Human whole blood was either stimulated with 40,000 U/ml of IFN-α for 15 minutes at 37ºC (solid line histogram) or was left unstimulated (dashed line histogram). The erythrocytes were lysed, and the leucocytes were fixed with 1× BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 15 minutes at 37ºC. The leucocytes were washed, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, washed, and then stained with BD Phosflow™R718 Mouse Anti-Stat4 (pY693) antibody (Cat. No.567631). The fluorescence histograms showing Stat4 (pY693) expression were derived from 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.

Flow cytometric analysis of Stat4 (pY693) expression in IFN-α treated human peripheral blood lymphocytes. Human whole blood was either stimulated with 40,000 U/ml of IFN-α for 15 minutes at 37ºC (solid line histogram) or was left unstimulated (dashed line histogram). The erythrocytes were lysed, and the leucocytes were fixed with 1× BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 15 minutes at 37ºC. The leucocytes were washed, permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes, washed, and then stained with BD Phosflow™R718 Mouse Anti-Stat4 (pY693) antibody (Cat. No.567631). The fluorescence histograms showing Stat4 (pY693) expression were derived from 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.

Product Details
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BD Phosflow™
Signal transducer and activator of transcription 4; SLEB11
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG2b, κ
Phosphorylated Human Stat4 (pY693)
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
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 CompBead to ensure that BD CompBeads are appropriate for your specific cellular application.

Product Notices

  1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  2. 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.
  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 refer to http://regdocs.bd.com to access safety data sheets (SDS).
  6. 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.
  7. Alexa Fluor™ is a trademark of Life Technologies Corporation.
  8. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
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Antibody Details
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38/p-Stat4

The Stat proteins function both as cytoplasmic signal transducers and as activators of transcription. Seven mammalian Stat proteins have been identified: Stat1-4, Stat5a, 5b, and Stat6.  Stat4 has been shown to play an important role in development of T helper cells, specifically the Th1 subset. Stat4 is activated by IL-12 and by type 1 interferons. Knockout mice supported the role that Stat4 plays in IL-12 signaling because lymphocytes from Stat 4-/- mice could neither differentiate into Th1 cells nor produce IFN-γ in response to treatment with IL-12. IFN-γ plays an important role in host defense. A key component in the activation of Stat4 is the phosphorylation on tyrosine and serine residues in response to IL-12 stimulation. IL-12 stimulation leads to the phosphorylation of Stat4 on tyrosine 693 and serine 721. Transcriptional activity of Stat4 has been shown to be significantly reduced when residues Y693 and S721 are mutated.

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.

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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
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Citations & References
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Development References (4)

  1. Cardone J, Le Friec G, Vantourout P, et al. Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells.. Nat Immunol. 2010; 11(9):862-71. (Clone-specific: Flow cytometry). View Reference
  2. Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene. 2002; 285:1-24. (Biology).
  3. Mowel WK, McCright SJ, Kotzin JJ, et al. Group 1 Innate Lymphoid Cell Lineage Identity Is Determined by a cis-Regulatory Element Marked by a Long Non-coding RNA.. Immunity. 2017; 47(3):435-449.e8. (Clone-specific: Flow cytometry). View Reference
  4. Visconti R, Gadina M, Chiariello M, et al. Importance of the MKK6/p38 pathway for interleukin-12−induced STAT4 serine phosphorylation and transcriptional activity. Blood. 2000; 96:1844-1852. (Biology).
View All (4) View Less
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.