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Two-color flow cytometric analysis of T-bet expression in human peripheral blood lymphocytes. Whole blood was treated with BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) to lyse erythrocytes and fix leucocytes. The leucocytes were permeabilized by treatment with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then washed and stained with BD Horizon™ BUV395 Mouse Anti-Human CD3 antibody (Cat. No. 563546) and with either BD Horizon™ BV480 Mouse IgG1, κ Isotype Control (Cat. No. 565652; Left Plot) or BD Horizon™ BV480 Mouse Anti-T-bet antibody (Cat. No. 568111/568165; Right Plot). The bivariate pseudocolor density plot showing the correlated expression of T-bet (or Ig Isotype control staining) was 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 Horizon™ BV480 Mouse Anti-T-bet
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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. This will ensure that BD® CompBeads are appropriate for your specific cellular application.
For optimal and reproducible results, BD Horizon Brilliant Stain Buffer should be used anytime BD Horizon Brilliant dyes are used in a multicolor flow cytometry panel. Fluorescent dye interactions may cause staining artifacts which may affect data interpretation. The BD Horizon Brilliant Stain Buffer was designed to minimize these interactions. When BD Horizon Brilliant Stain Buffer is used in in the multicolor panel, it should also be used in the corresponding compensation controls for all dyes to achieve the most accurate compensation. For the most accurate compensation, compensation controls created with either cells or beads should be exposed to BD Horizon Brilliant Stain Buffer for the same length of time as the corresponding multicolor panel. More information can be found in the Technical Data Sheet of the BD Horizon Brilliant Stain Buffer (Cat. No. 563794/566349) or the BD Horizon Brilliant Stain Buffer Plus (Cat. No. 566385/568264).
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Companion Products
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.
Development References (10)
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Bando JK, Gilfillan S, Di Luccia B, et al. ILC2s are the predominant source of intestinal ILC-derived IL-10.. J Exp Med. 2020; 217(2):e20191520. (Clone-specific: Flow cytometry). View Reference
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Cella M, Gamini R, Sécca C, et al. Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.. Nat Immunol. 2019; 20(8):980-991. (Clone-specific: Flow cytometry). View Reference
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Kawabe T, Yi J, Kawajiri A, et al. Requirements for the differentiation of innate T-bethigh memory-phenotype CD4+ T lymphocytes under steady state.. Nat Commun. 2020; 11(1):3366. (Clone-specific: Flow cytometry). View Reference
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Li S, Sullivan NL, Rouphael N, et al. Metabolic Phenotypes of Response to Vaccination in Humans.. Cell. 2017; 169(5):862-877.e17. (Clone-specific: Flow cytometry). View Reference
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Patel PS, King RG, Kearney JF. Pulmonary α-1,3-Glucan-Specific IgA-Secreting B Cells Suppress the Development of Cockroach Allergy.. J Immunol. 2016; 197(8):3175-3187. (Clone-specific: Flow cytometry). View Reference
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Peng SL. The T-box transcription factor T-bet in immunity and autoimmunity. Cell Mol Immunol. 2006; 3(2):87-95. (Biology). View Reference
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Ponzetta A, Carriero R, Carnevale S, et al. Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors.. Cell. 2019; 178(2):346-360.e24. (Clone-specific: Flow cytometry). View Reference
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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. (Clone-specific: Flow cytometry). View Reference
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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). View Reference
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Trotta E, Bessette PH, Silveria SL, et al. A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.. Nat Med. 2018; 24(7):1005-1014. (Clone-specific: Flow cytometry). View Reference
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