Skip to main content Skip to navigation
PE Mouse anti-T-bet
PE Mouse anti-T-bet

Flow cytometric analysis of T-bet expression by human peripheral blood lymphocytes.

     Left and Middle Left Panels: Whole blood was treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) to lyse erythrocytes and fix leukocytes. Cells were permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885; Left Panel) or Perm Buffer III (Cat. No. 558050; Middle Left Panel). Cells were stained with Alexa Fluor® 647 Mouse Anti-Human CD3 (Cat. No. 557706) and PE Mouse anti-T-Bet (Cat. No. 561268) antibodies. Two-color dot plots showing CD3 versus T-bet expression for lymphocytes were generated using a BD FACSCalibur™ Flow Cytometer.

     Middle Right Panel: Whole blood was treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049). Cells were permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), washed, and stained with PE Mouse anti-T-Bet (Cat. No. 561268), FITC Mouse Anti-Human CD45RO (Cat. No. 555492), APC Mouse Anti-Human CD45RA (Cat. No. 550855), BD Horizon™ V450 Mouse Anti-Human CD3 (Cat. No. 560365), and PerCP Mouse Anti-Human CD4 (Cat. No. 347324) antibodies. Two-color contour plots showing CD45RA (Top Plot) or CD45RO (Bottom Plot) versus T-bet expression patterns for CD3+CD4+ lymphocytes were generated using a BD FACSCanto™ II Flow Cytometer.

     Right Panel: Human peripheral blood mononuclear cells were polarized in culture to generate Th1- (shaded histogram) or Th2-like (open histogram) cells. The cells were restimulated with PMA (Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634; 1 ug/ml). The cells were then fixed, permeabilized, and stained with PE Mouse anti-T-Bet (Cat. No. 561268), V450 Mouse Anti-Human CD3 (Cat. No. 560366) and PerCP-Cy™5.5 Mouse Anti-Human CD4 (Cat. No. 560650) using BD Biosciences Protocol for Intracellular Cytokine Staining. The histograms for CD3+CD4+ lymphocytes expressing T-bet were generated using a BD FACSCanto™ II Flow Cytometer.

Flow cytometric analysis of T-bet expression by human peripheral blood lymphocytes.

     Left and Middle Left Panels: Whole blood was treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) to lyse erythrocytes and fix leukocytes. Cells were permeabilized with BD Phosflow™ Perm/Wash Buffer I (Cat. No. 557885; Left Panel) or Perm Buffer III (Cat. No. 558050; Middle Left Panel). Cells were stained with Alexa Fluor® 647 Mouse Anti-Human CD3 (Cat. No. 557706) and PE Mouse anti-T-Bet (Cat. No. 561268) antibodies. Two-color dot plots showing CD3 versus T-bet expression for lymphocytes were generated using a BD FACSCalibur™ Flow Cytometer.

     Middle Right Panel: Whole blood was treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049). Cells were permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050), washed, and stained with PE Mouse anti-T-Bet (Cat. No. 561268), FITC Mouse Anti-Human CD45RO (Cat. No. 555492), APC Mouse Anti-Human CD45RA (Cat. No. 550855), BD Horizon™ V450 Mouse Anti-Human CD3 (Cat. No. 560365), and PerCP Mouse Anti-Human CD4 (Cat. No. 347324) antibodies. Two-color contour plots showing CD45RA (Top Plot) or CD45RO (Bottom Plot) versus T-bet expression patterns for CD3+CD4+ lymphocytes were generated using a BD FACSCanto™ II Flow Cytometer.

     Right Panel: Human peripheral blood mononuclear cells were polarized in culture to generate Th1- (shaded histogram) or Th2-like (open histogram) cells. The cells were restimulated with PMA (Sigma Cat. No. P-8139) and Ionomycin (Sigma Cat. No. I-0634; 1 ug/ml). The cells were then fixed, permeabilized, and stained with PE Mouse anti-T-Bet (Cat. No. 561268), V450 Mouse Anti-Human CD3 (Cat. No. 560366) and PerCP-Cy™5.5 Mouse Anti-Human CD4 (Cat. No. 560650) using BD Biosciences Protocol for Intracellular Cytokine Staining. The histograms for CD3+CD4+ lymphocytes expressing T-bet were generated using a BD FACSCanto™ II Flow Cytometer.

Product Details
Down Arrow Up Arrow


BD Pharmingen™
T-box expressed in T cells; TBX21; T-box 21; TBLYM
Human (QC Testing), Mouse (Tested in Development)
Mouse IgG1, κ
Human T-bet Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_10564071
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 with R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Recommended Assay Procedures

Immunofluorescent staining of intracellulat T-bet with the PE O4-46 Mouse Anti-T-bet monoclonal antibody (Cat. No. 561268) is compatible with various Fixatives and Permeabilization Buffers from BD Biosciences (please see Suggested Companion Products).  For more information on the BD Pharmingen™ Transcription Factor Buffer Set please refer to http://www.bdbiosciences.com/documents/BD_Pharmigen_TranscriptFactorBuffer_DS.pdf

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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. 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).
  6. An isotype control should be used at the same concentration as the antibody of interest.
  7. This product may be covered by US Patent No. 7,365,169.
  8. Limited Use License: The buyer (a) shall not sell or otherwise transfer this product to any third party, (b) shall use this product only for its internal, non-clinical research use, (c) shall not use this product for Commercial Purposes without a commercial license from Harvard (and if the buyer is interested in a commercial license, it should contact Harvard’s Office of Technology Development at 1350 Massachusetts Avenue, Holyoke Center, Suite 727, Cambridge, MA 02138, 617-495-3067), (d) shall use this product in compliance with all applicable laws and regulations, including, without limitation, applicable human health and animal welfare laws and regulations, (e) may transfer information or materials made through the use of this product to a scientific collaborator only if such transfer is not for any Commercial Purpose and such collaborator agrees in writing not to transfer such materials to any third party and to use such transferred materials and/or information solely for internal, non-clinical research and not for Commercial Purposes, (f) acknowledges that this product has not been approved for use in humans by the U.S. Food and Drug Administration or any other regulatory body and may not be used in humans and (g) shall indemnify, defend and hold harmless Becton Dickinson and Company and Harvard from and against all damages, losses, expenses (including reasonable attorneys’ fees), claims, demands, suits and other actions in any way arising from the buyer’s use, storage or disposal of this product.
  9. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  10. Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
561268 Rev. 2
Antibody Details
Down Arrow Up Arrow
O4-46

The O4-46  monoclonal antibody specifically binds to human and mouse T-bet. T-bet (T-box gene expressed in T cells) is a master regulatory transcription factor that is also known as TBX21 (T-box21) and TBLYM (T-box transcription factor, expressed in lymphocytes). Human (535 amino acids; 58.3 kDa predicted molecular mass) and mouse (530 amino acids; 57.7 kDa) T-bet proteins are encoded by the human TBX21 (chromosome 17) and mouse Tbx21 (chromosome 11) genes. The human and mouse T-bet protein amino acid sequences are 88% homologous. Human and mouse T-bet proteins share a highly conserved (98% homologous amino acid sequences) T-box protein domain that is centrally located and mediates binding to DNA. T-bet is expressed by and activates transcriptional activities within hemotopoietic cells including stem cells,  NK and NKT cells and subsets of thymocytes, primed/activated CD4+ T cells, CD8+ T cells and γδ T cells, B cells, and dendritic cells. Interferon-gamma (IFN-γ), interleukin-27 (IL-27), and IL-12 act on peripheral antigen-triggered (TCR-signaling) T cells to increase T-bet expression. With respect to T helper lymphocytes, T-bet directs the differentiation of naïve CD4+ precursor T cells to become Th1-like effector and memory cells. T-bet accomplishes this by activating Th1 genetic programs (including epigenetic modifications) while repressing opposing T helper subset programs. T-bet controls the upregulated expression of the Th1 signature cytokine, IFN-γ, the IL-12Rβ2 subunit and the Runx3 transcription factor and can repress the function of other transcriptional regulators, such as GATA-3 (master regulator of Th2 development) and the expression of other cytokines including IL-2, IL-4 and IL-5.

561268 Rev. 2
Format Details
Down Arrow Up Arrow
PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
561268 Rev.2
Citations & References
Down Arrow Up Arrow

Development References (10)

  1. Hibbert L, Pflanz S, De Waal Malefyt R, Kastelein RA. IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells. J Interferon Cytokine Res. 2003; 23(9):513-522. (Biology). View Reference
  2. Hwang ES, Hong JH, Glimcher LH. IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. J Exp Med. 2005; 202(9):1289-1300. (Biology: Western blot). View Reference
  3. Hwang ES, Szabo SJ, Schwartzberg PL, Glimcher LH. T helper cell fate specified by kinase-mediated interaction of T-bet with GATA-3. Science. 2005; 307(5780):430-433. (Biology: Western blot). View Reference
  4. Lugo-Villarino G, Maldonado-Lopez R, Possemato R, Penaranda C, Glimcher LH. T-bet is required for optimal production of IFN-gamma and antigen-specific T cell activation by dendritic cells. Proc Natl Acad Sci U S A. 2003; 100(13):7749-7754. (Biology). View Reference
  5. Peng SL, Szabo SJ, Glimcher LH. T-bet regulates IgG class switching and pathogenic autoantibody production. Proc Natl Acad Sci U S A. 2002; 99(8):5545-5550. (Biology). View Reference
  6. Peng SL. The T-box transcription factor T-bet in immunity and autoimmunity. Cell Mol Immunol. 2006; 3(2):87-95. (Biology). View Reference
  7. Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell. 2000; 100(6):655-669. (Biology: Western blot). View Reference
  8. Takeda A, Hamano S, Yamanaka A, et al. Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment. J Immunol. 2003; 170(10):4886-4890. (Biology). View Reference
  9. Townsend MJ, Weinmann AS, Matsuda JL, et al. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity. 2004; 20(4):477-494. (Biology). View Reference
  10. Zhang WX, Yang SY. Cloning and characterization of a new member of the T-box gene family. Genomics. 2000; 70(1):41-48. (Biology). View Reference
View All (10) View Less
561268 Rev. 2

Please refer to Support Documents for Quality Certificates


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.