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Alexa Fluor™ 647 Mouse Anti-RUNX3
Alexa Fluor™ 647 Mouse Anti-RUNX3

Multicolor flow cytometric analysis of RUNX3 expression in human peripheral blood cells and mouse lymph node cells.

Panel 1. Human peripheral blood mononuclear cells (PBMC) were stained with BD Horizon™ BUV395 Mouse Anti-Human CD8α (Cat. No. 563796), PE Mouse Anti-Human CD3 (Cat. No. 555333/561808/561809) and FITC Mouse Anti-Human CD56 (NCAM-1) (Cat. No. 562794) antibodies. The cells were fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725) and stained with either Alexa Fluor™ 647 Mouse IgG1 κ Isotype Control (Cat. No. 565571) or Alexa Fluor™ 647 Mouse Anti-RUNX3 antibody (Cat. No. 565741). The bivariate pseudocolor density plots on the left show the correlated expression of Ig Isotype control staining (Upper Left Plot) or RUNX3 (Bottom Left Plot) versus CD8α for gated events with the light scattering characteristics of intact lymphocytes. Intact cells were also gated as indicated (Upper Right Plot) and used for the generation of the overlaid dot plot figure (Bottom Right Plot) showing the correlated expression of RUNX3 versus CD3 for the CD8+CD56+ (blue), CD8+CD56- (green), and CD8-CD56+ (red) cell subsets.

Panel 2. C57BL/6 mouse lymph node cells (LNC) were stained with FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553047/553046/561835) and then fixed and permeabilized (as described above), followed by staining with either Alexa Fluor™ 647 Mouse IgG1, κ Isotype Control (Upper Plot) or Alexa Fluor™ 647 Mouse Anti-RUNX3 antibody (Bottom Plot). The bivariate pseudocolor density plots showing the correlated expression of RUNX3 (or Ig Isotype control staining) versus CD4 were derived from gated events with the light-scatter characteristics of intact lymphocytes.

Flow cytometry and data analysis was performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.

Multicolor flow cytometric analysis of RUNX3 expression in human peripheral blood cells and mouse lymph node cells.

Panel 1. Human peripheral blood mononuclear cells (PBMC) were stained with BD Horizon™ BUV395 Mouse Anti-Human CD8α (Cat. No. 563796), PE Mouse Anti-Human CD3 (Cat. No. 555333/561808/561809) and FITC Mouse Anti-Human CD56 (NCAM-1) (Cat. No. 562794) antibodies. The cells were fixed and permeabilized with the BD Pharmingen™ Transcription Factor Buffer Set (Cat. No. 562574/562725) and stained with either Alexa Fluor™ 647 Mouse IgG1 κ Isotype Control (Cat. No. 565571) or Alexa Fluor™ 647 Mouse Anti-RUNX3 antibody (Cat. No. 565741). The bivariate pseudocolor density plots on the left show the correlated expression of Ig Isotype control staining (Upper Left Plot) or RUNX3 (Bottom Left Plot) versus CD8α for gated events with the light scattering characteristics of intact lymphocytes. Intact cells were also gated as indicated (Upper Right Plot) and used for the generation of the overlaid dot plot figure (Bottom Right Plot) showing the correlated expression of RUNX3 versus CD3 for the CD8+CD56+ (blue), CD8+CD56- (green), and CD8-CD56+ (red) cell subsets.

Panel 2. C57BL/6 mouse lymph node cells (LNC) were stained with FITC Rat Anti-Mouse CD4 antibody (Cat. No. 553047/553046/561835) and then fixed and permeabilized (as described above), followed by staining with either Alexa Fluor™ 647 Mouse IgG1, κ Isotype Control (Upper Plot) or Alexa Fluor™ 647 Mouse Anti-RUNX3 antibody (Bottom Plot). The bivariate pseudocolor density plots showing the correlated expression of RUNX3 (or Ig Isotype control staining) versus CD4 were derived from gated events with the light-scatter characteristics of intact lymphocytes.

Flow cytometry and data analysis was performed using a BD LSRFortessa™ Flow Cytometer System and FlowJo™ software.

Product Details
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BD Pharmingen™
AML2; AML-2; CBFA3; PEBP2aC; PEBP2A3
Human (QC Testing), Mouse (Tested in Development)
Mouse BALB/c IgG1, κ
Human RUNX3 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
Aqueous buffered solution containing BSA and ≤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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  6. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
  7. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  8. This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
  9. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  10. Alexa Fluor™ is a trademark of Life Technologies Corporation.
  11. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
565741 Rev. 1
Antibody Details
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R3-5G4

The R3-5G4 monoclonal antibody specifically binds to Runt-related transcription factor 3 (RUNX3) which is also known as Acute myeloid leukemia 2 protein (AML2), Core-binding factor subunit alpha-3 (CBFA3), and Polyomavirus enhancer-binding protein 2 alpha C subunit (PEBP2aC).  RUNX3 is a member of the RUNX transcription factor family which includes RUNX1-3. RUNX3 is a key regulator of gene expression related to the development and differentiation of cells within the nervous and immune systems. In the immune system, RUNX3 is particularly involved in the commitment of CD8+ T cells in the thymus. RUNX3 is highly expressed and essential for the cytotoxic functions of NK cells, peripheral CD8+T cells and CD4+CD8αα intraepithelial lymphocytes in the gut. RUNX3 also plays a role in the differentiation and effector functions of Th1 cells. RUNX3 is activated downstream of the TGF-β signaling pathway and can play a role in tumor suppression. Aberrant expression of RUNX3 has been associated with tumorigenesis including the development of gastric and other cancers.

565741 Rev. 1
Format Details
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Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
565741 Rev.1
Citations & References
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Development References (6)

  1. Chuang LS, Ito K, Ito Y. RUNX family: Regulation and diversification of roles through interacting proteins. Int J Cancer. 2013; 132(6):1260-1271. (Biology). View Reference
  2. Djuretic IM, Cruz-Guilloty F, Rao A. Regulation of gene expression in peripheral T cells by Runx transcription factors. Adv Immunol. 2009; 104(1):23. (Biology). View Reference
  3. Ito K, Liu Q, Salto-Tellez M, et al. RUNX3, a novel tumor suppressor, is frequently inactivated in gastric cancer by protein mislocalization. Cancer Res. 2005; 65(17):7743-7750. (Immunogen: Western blot). View Reference
  4. Lotem J, Levanon D, Negreanu V, Leshkowitz D, Friedlander G, Groner Y. Runx3-mediated transcriptional program in cytotoxic lymphocytes. PLoS ONE. 2013; 8(11):e80467. (Biology). View Reference
  5. Reis BS, Rogoz A, Costa-Pinto FA, Taniuchi I, Mucida D. Mutual expression of the transcription factors Runx3 and ThPOK regulates intestinal CD4(+) T cell immunity. Nat Immunol. 2013; 14(3):271-280. (Biology). View Reference
  6. Setoguchi R, Tachibana M, Naoe Y, et al. Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell devel. Science. 2008; 19(5864):822-825. (Biology). View Reference
View All (6) View Less
565741 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.