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Anti t bet

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Anti-T-bet is a laboratory reagent used in research applications. It is an antibody that specifically binds to the T-bet transcription factor, which plays a key role in the development and function of T helper 1 (Th1) cells. This reagent can be utilized in various immunological assays and techniques to study T-cell biology and differentiation.

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Lab products found in correlation

Anti rorγt, by Thermo Fisher Scientific (7 mentions) Anti il 17a, by Thermo Fisher Scientific (3 mentions) Anti foxp3, by Thermo Fisher Scientific (3 mentions) Anti gata3, by Thermo Fisher Scientific (3 mentions) Facsaria 3, by BD (2 mentions) Ionomycin, by Merck Group (2 mentions) Anti il 10, by Thermo Fisher Scientific (2 mentions) Anti cd16 cd32, by Thermo Fisher Scientific (2 mentions) 7 aad viability dye, by Thermo Fisher Scientific (2 mentions) Anti cd45, by BioLegend (2 mentions) Anti cd27, by BioLegend (2 mentions) Anti cxcr4, by BioLegend (2 mentions) Anti eomes, by Thermo Fisher Scientific (2 mentions) Anti nkp46, by Thermo Fisher Scientific (2 mentions) Anti α4β7, by BioLegend (2 mentions) Anti cd49b, by Thermo Fisher Scientific (2 mentions) Anti il 17, by BioLegend (2 mentions) Anti rat igg1k isotype control, by BioLegend (2 mentions) Anti streptavidin fluorochrome conjugates, by BioLegend (2 mentions) Anti flt3, by BioLegend (2 mentions)

Spelling variants of this product

T-bet (36 citations) Anti-T-bet (4B10) (8 citations) Anti-T-bet-PE-Cy7 (7 citations) PEcy7-conjugated anti-T-bet (3 citations) Anti-T-bet (eBio4B10) (3 citations) Anti- mouse T-bet PE (2 citations) PE-conjugated anti-mouse T-bet (2 citations) Anti-human/mouse T-bet PE (2 citations) Anti-T-Bet (clone 4B10) (2 citations) PECy7 anti-T-bet (1 citations)

17 protocols using anti t bet

1

Intracellular Cytokine and Phospho-flow Analysis

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For intracellular cytokine staining, murine cells were restimulated after 72 h of culture with PMA (50 ng ml−1), Ionomycin (1 µg ml−1, both from Sigma-Aldrich) and brefeldin A (5 µg ml−1; Biolegend) for 4 h and fixed with 2% para-formaldehyde. Staining of transcription factors was performed without restimulation using the FOXP3/Fixation-Kit (eBioscience, 00–5521–00). Following antibodies were used for murine cell-analysis: anti-CD8a (eBiosciences, 53–6.7), anti-CD44 (eBiosciences, IM7), anti-CD62L (eBiosciences, MEL-14), anti-IFN-y (Biolegend, XMG1.2), anti-IL-17A (eBiosciences, eBio17B7), anti-RORyt (eBiosciences, B2D) or anti-T-BET (eBiosciences, eBio4B10). The cells were measured on FACSCalibur or FACSAria™ III (both BD) and analyzed with FlowJo Software (FlowJo LLC). For phospho-flow the cells were harvested after 48 h of culture, rested for 4 h and treated with 100U/ml rhIL-2 for 2 h, fixed and permeabilised using Lyse/Fix-Buffer (557870, BD) and Perm-BufferIII (558050, BD) then stained with anti-P-STAT5Y694 (eBiosciences, SRBCZX), anti-P-AKTS473 (Cell Signaling, D9E), anti-P-AKTY308 (Cell Signaling, D25E6), anti-P-FOXO1/3a (Cell Signaling, #9464) or anti-P-S6S235/236 (Cell Signaling, D57.2.2E).
Lückel C., Picard F., Raifer H., Campos Carrascosa L., Guralnik A., Zhang Y., Klein M., Bittner S., Steffen F., Moos S., Marini F., Gloury R., Kurschus F.C., Chao Y.Y., Bertrams W., Sexl V., Schmeck B., Bonetti L., Grusdat M., Lohoff M., Zielinski C.E., Zipp F., Kallies A., Brenner D., Berger M., Bopp T., Tackenberg B, & Huber M. (2019). IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis. Nature Communications, 10, 5722.
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2

Profiling Lung Tissue Protein Expression

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The proteins of the lung tissues in mice were extracted for Western blot analysis as described previously.20 Rat monoclonal antibodies of anti‐T‐bet (eBioscience) (1:250 dilution), anti‐GATA‐3 (Cell Signaling Technology) (1:1000 dilution), anti‐RORγ(t) (eBioscience) (1:100 dilution) and GAPDH (Cell Signaling Technology) (1:1000 dilution) were used as the primary antibodies. HRP‐conjugated anti‐rat IgG (Beyotime Biotechnology) was also utilized. ECL chemiluminescence kit was used for chemiluminescent detection followed by image analysis.
Zhang W., Li L., Zheng Y., Xue F., Yu M., Ma Y., Dong L., Shan Z., Feng D., Wang T, & Wang X. (2019). Schistosoma japonicum peptide SJMHE1 suppresses airway inflammation of allergic asthma in mice. Journal of Cellular and Molecular Medicine, 23(11), 7819-7829.
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3

Multiparametric Flow Cytometry Analysis of Immune Cell Phenotypes

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The following antibodies were used: anti-mouse TCR-β (H57-597) APC or Pacific Blue, PBS57 loaded CD1d tetramer APC or Pacific Blue, anti-mouse CD4 (GK1.5) PerCp-Cy5.5, anti-mouse CD8 (53-6.7) Am-cyan, anti-mouse NK1.1 (PK-136) PE-Cy7, anti-mouse CD44 (IM7) PerCp-Cy5.5, anti-mouse CD69 (H1-2F3) PE-Cy7, anti-mouse CD62L (MEL-14) eVolve605, anti-mouse IFN-γ (XMG1.2) FITC or APC, anti-mouse IL-4 (11B11) PE-Cy7, anti-mouse IL-17 (TC11-18H10) APC-eFluor780, anti-T-bet (eBio4B10) FITC, anti-RORγt (AFKJS-9) Pacific Blue, anti-GATA3 (L50-823) APC, and anti-PLZF (Mags-21F7) PE (all from eBioscience). Dead cells were excluded by staining with 1 µg/ml propidium iodide (Sigma-Aldrich). To measure intracellular cytokines, cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, Sigma-Aldrich) and ionomycin (1.5 µM, Sigma-Aldrich) in the presence of Monensin (3 µM, Sigma-Aldrich), permeabilized using Cytofix/Cytoperm Plus (BD), then stained with the appropriate antibodies. Transcription factor staining to identify committed cells was performed using the Foxp3/transcription factor staining kit (eBioscience) and intranuclear staining for T-bet, RORγt, GATA3 and PLZF. Data were acquired on a FACS Canto II (BD) and analyzed using FlowJo (TreeStar software ver. 9.9).
Kim Y.H., Kumar A., Chang C.H, & Pyaram K. (2017). Reactive Oxygen Species Regulate the Inflammatory Function of NKT Cells through Promyelocytic Leukemia Zinc Finger. Journal of immunology (Baltimore, Md. : 1950), 199(10), 3478-3487.
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4

Multiparametric Flow Cytometry of T-Cells

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Five replicates of splenocytes (105) were plated in 200 µl of medium supplemented with 0.05 µg/ml PMA (Sigma-Aldrich), 0.001 µg/ml ionomycin calcium salt (Sigma-Aldrich) and 0.2 µl BD GolgiPlug (brefeldin A, BD Pharmingen). As control, the cells were stimulated with PMA and Ionomycin without GolgiPlug. After 4 h of incubation at 37°C and 5% CO2, 100 µl of the supernatant was frozen at −20°C for ELISA. The cells were first stained with anti-CD8α and anti-CD4 before following the instruction for cytokines’ intracellular staining with a mix of anti-IL-2, anti-IL-4, anti-IL-10, anti-IL-17, anti-IL-22 and anti-IFN-γ, or intracellular staining for transcription factors with a mix of anti-Foxp3, anti-T-bet, anti-ROR-γt and anti-Gata-3 (eBioscience). The staining of intracellular cytokines and enzymes of CD8+ T-cells was done with a mix of anti-IL-2, anti-IFN-γ, anti-granzyme B and anti-perforin (reagents and isotypes from eBioscience). For FACS analysis, cells were resuspended in 50 µl PBS (1x).
Floderer M., Prchal-Murphy M, & Vizzardelli C. (2014). Dendritic Cell-Secreted Lipocalin2 Induces CD8+ T-Cell Apoptosis, Contributes to T-Cell Priming and Leads to a TH1 Phenotype. PLoS ONE, 9(7), e101881.
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5

Immunophenotyping of T-cell subsets

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FACS staining was conducted as described29 (link) using the following antibodies: anti-mouse CD4 (L3T4, BD), anti-mouse TCR (553169, BD), anti-FoxP3 (17–5773–82, eBioscience), anti-T-bet (25–5825–82, eBioscience), anti-RORγt (17–6981–82, eBioscience), anti-CD69 (15–0691–82, eBioscience), pZAP-70 (557817, BD), anti-Ki-67 (612472, BD), and anti-ADAM12 7B8 (in-house).
Cells were acquired using FACSDiva after the exclusion of duplets. Dead cells were discriminated in all stains using the LIVE/DEAD Fixable Dead Cell Stain Kit at 405-nm excitation (L34955, Invitrogen). FlowJo 10.5.3 (Flowjo, LLC) was used for further analysis.
Liu Y., Bockermann R., Hadi M., Safari I., Carrion B., Kveiborg M, & Issazadeh-Navikas S. (2020). ADAM12 is a costimulatory molecule that determines Th1 cell fate and mediates tissue inflammation. Cellular and Molecular Immunology, 18(8), 1904-1919.
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6

Multiparametric Flow Cytometry Immune Profiling

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Cell suspensions were stained with: anti-CD45 (30-F11); anti-CD45.1 (A20); anti-CD45.2 (104); anti-CD11c (N418); anti-CD11b (Mi/70); anti-CD127 (IL7Rα; A7R34); anti-CD27(LG.7F9); anti-CD8α (53-6.7); anti-CD19 (eBio1D3); anti-CXCR4(L276F12); anti-NK1.1 (PK136); anti-CD3ε (eBio500A2); anti-TER119 (TER-119); anti-Gr1 (RB6-8C5); anti-CD4 (RM4-5); anti-CD25 (PC61); anti-CD117 (c-Kit; 2B8); anti-CD90.2 (Thy1.2; 53-2.1); anti-TCRβ (H57-595); anti-TCRγδ (GL3); anti-B220 (RA3-6B2); anti-KLRG1 (2F1/KLRG1); anti-Ly-6A/E (Sca1; D7); anti-CCR9 (CW-1.2); anti-IL-17 (TC11-18H10.1); anti-rat IgG1k isotype control (RTK2071); anti-streptavidin fluorochrome conjugates from Biolegend; anti-α4β7 (DATK32); anti-Flt3 (A2F10); anti-NKp46 (29A1.4); anti-CD49b (DX5); anti-Ki67 (SolA15); anti-rat IgG2ak isotype control (eBR2a); anti-IL-22 (1H8PWSR); anti-rat IgG1k isotype control (eBRG1); anti-EOMES (Dan11mag); anti-Tbet (eBio4B10); anti-FOPX3 (FJK-16s); anti-GATA3 (TWAJ); anti-CD16/CD32 (93); 7AAD viability dye from eBiosciences; anti-CD196 (CCR6; 140706) from BD Biosciences; anti-RORγt (Q31-378) and anti-mouse IgG2ak isotype control (G155-178) from BD Pharmingen. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit was purchased from Invitrogen.
Godinho-Silva C., Domingues R.G., Rendas M., Raposo B., Ribeiro H., Alves da Silva J., Vieira A., Costa R.M., Morais-Barbosa N.L., Carvalho T, & Veiga-Fernandes H. (2019). Light-entrained and brain-tuned circadian circuits regulate ILC3 and gut homeostasis. Nature, 574(7777), 254-258.
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7

Multiparametric Profiling of T Cell Subsets

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eFluor660-anti–IL-21, Alexa Fluor 488–anti–IL-10, PerCP-Cy5.5-anti–IFN-γ, FITC-anti-CD45RA, PE-anti-ICOS, and anti-Tbet, and biotin–PD-1 were from eBioscience. Alexa Fluor 488–anti-GATA3, Alexa Fluor 647–anti-CXCR5, anti-pSTAT4, anti-pSTAT5, anti-pSTAT6, APC-anti-CD10, APC-Cy7-anti-CD4, BV605-anti-IgG, BV421-CD40L, BV711-anti–IL-2, PE-anti-pSTAT1, anti-RORγt, and Bcl-6, PE-Cy7-anti-CD25, and anti-CD27, PerCP-Cy5.5-anti-CD127, anti-pSTAT3, and anti-Tbet, SA-PerCpCy5.5, and IFN-γ were obtained from BD. Pacific Blue–anti-CD20 and SA-BV605 were purchased from BioLegend. Recombinant human IL-12 was purchased from R&D Systems. TGF-β, IL-1β, IL-6, IL-21, and IL-23 were obtained from PeproTech. PGE2 was purchased from Sigma-Aldrich. Human IL-4 and IL-10 were provided by R. de Waal Malefyt (DNAX Research Institute, Palo Alto, CA).
Ma C.S., Wong N., Rao G., Nguyen A., Avery D.T., Payne K., Torpy J., O’Young P., Deenick E., Bustamante J., Puel A., Okada S., Kobayashi M., Martinez-Barricarte R., Elliott M., Sebnem Kilic S., El Baghdadi J., Minegishi Y., Bousfiha A., Robertson N., Hambleton S., Arkwright P.D., French M., Blincoe A.K., Hsu P., Campbell D.E., Stormon M.O., Wong M., Adelstein S., Fulcher D.A., Cook M.C., Stepensky P., Boztug K., Beier R., Ikincioğullari A., Ziegler J.B., Gray P., Picard C., Boisson-Dupuis S., Phan T.G., Grimbacher B., Warnatz K., Holland S.M., Uzel G., Casanova J.L, & Tangye S.G. (2016). Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets. The Journal of Experimental Medicine, 213(8), 1589-1608.
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8

Stimulation and Cytokine Profiling of T Cells

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T cells purified by cell sorting were stimulated with Leukocyte Activation Cocktail, with GolgiPlus™ (BD Pharmingen) for 6 h at 37°C under 5% CO2 before being stained with following antibodies alone or in combinations: anti-IL-17A (eBioscience), anti-IL-17F, anti-IL-22, anti-IL-6, anti-CCL20 and anti-IFNγ (R&D Systems) by using Cytofix/CytoPerm Plus kit (BD Pharmingen) or anti-RORγt, anti-FOXP3, anti-T-bet or anti-GATA3 using Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instructions. All flow cytometry was done using FACSCaliber, LSR II System or Fortessa flow cytometers (BD Biosciences), and the data were subsequently analyzed using FlowJO software (TreeStar).
Singh S.P., Parween F., Edara N., Zhang H.H., Chen J., Otaizo-Carrasquero F., Cheng D., Oppenheim N.A., Ransier A., Zhu W., Shamsaddini A., Gardina P.J., Darko S.W., Singh T.P., Douek D.C., Myers T.G, & Farber J.M. (2023). Human CCR6+ Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity. Journal of immunology (Baltimore, Md. : 1950), 210(11), 1700-1716.
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9

Intracellular Transcription Factor Detection

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To detect intracellular transcription factors, anti–mouse Foxp3 staining kits (eBioscience) were used after appropriate surface staining. Staining was performed according to the manufacturer’s instructions. In brief, cells were resuspended in fixation/permeabilization buffer and incubated at 4°C, in the dark for 1 h. Cells were then washed with Perm/Wash buffer (eBioscience) before being labeled with anti-PLZF (eBioscience), anti–T-Bet (eBioscience), and anti-RORγt (eBioscience). This approach was also used for intracellular transcription factor and cytokine costaining.
Rahimpour A., Koay H.F., Enders A., Clanchy R., Eckle S.B., Meehan B., Chen Z., Whittle B., Liu L., Fairlie D.P., Goodnow C.C., McCluskey J., Rossjohn J., Uldrich A.P., Pellicci D.G, & Godfrey D.I. (2015). Identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant T cells using MR1 tetramers. The Journal of Experimental Medicine, 212(7), 1095-1108.
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10

Cytokine Production Analysis by Flow Cytometry

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For analysis of cytokine production, cells were restimulated with 20 nM phorbol 12-myristate 13-acetate (PMA) (Calbiochem) and 1 μM ionomycin (Invitrogen) in the presence of 5 μM brefeldin A for 6 h. Cells were stained with the following antibodies against cell surface markers (all from eBioscience unless otherwise indicated): anti-CD4 (GK1.5), anti-CD45.2 (104), anti-CD8 (53-6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-Gr-1 (RB6-8C5), anti-TGF-β (TW7-16B4; Biolegend). Cells were fixed and permeabilized with either IC Fix Buffer (eBioscience) for intracellular cytokine staining or Foxp3 Fix Buffer (BioLegend) for Foxp3 staining using the following antibodies (all from eBioscience): anti-IFN-γ (XMG1.2), anti-IL-17 (eBio17B7), anti-GM-CSF (MP1-22E9), anti-IL-10 (JES5-16E3), anti-RORγt (B2D), anti-T-bet (eBio4B10) and anti-Foxp3 (FJK-16s). Cells were analyzed using a LSRII flow cytometer (BD Biosciences) and FlowJo software (Tree Star).
Kaufmann U., Shaw P.J., Kozhaya L., Subramanian R., Gaida K., Unutmaz D., McBride H.J, & Feske S. (2015). Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function. Journal of immunology (Baltimore, Md. : 1950), 196(2), 573-585.
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