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Anti tbet 4b10

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Anti-Tbet (4B10) is a mouse monoclonal antibody that recognizes the human T-box transcription factor T-bet. T-bet is a key transcription factor involved in the differentiation and function of T helper 1 (Th1) cells. The Anti-Tbet (4B10) antibody can be used for the detection and analysis of T-bet expression in various applications, such as flow cytometry, immunohistochemistry, and Western blotting.

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Lab products found in correlation

Anti cd4 gk1, by BioLegend (4 mentions) Anti ifn γ xmg1, by BioLegend (3 mentions) Anti rorγt q31 378, by BD (3 mentions) Anti cd69, by BioLegend (2 mentions) Anti cd28, by BioLegend (2 mentions) Anti vista mih63, by BioLegend (2 mentions) Anti cd5, by BioLegend (2 mentions) Facscalibur, by BD (2 mentions) Anti cd45.2 104, by BioLegend (2 mentions) Anti cd8a 53 6, by BioLegend (2 mentions) Anti cd11b m1 70, by BioLegend (2 mentions) Anti cd25 3c7, by BioLegend (2 mentions) Anti foxp3 mf14, by Thermo Fisher Scientific (2 mentions) Anti cd71 r17 217, by BioXCell (2 mentions) Depletion c2f2, by BD (2 mentions) Anti cd45 i3 2, by BioLegend (2 mentions) Anti cd90.1 16 10a1, by BioLegend (2 mentions) Anti gzmb gl1, by BioLegend (2 mentions) Anti cxcr5 l138d7, by BioLegend (2 mentions) Anti mouse human cd44 im7, by BioLegend (2 mentions)

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Anti-Tbet (clone 4B10, (6 citations)

13 protocols using anti tbet 4b10

1

Flow cytometry analysis of splenic immune cells

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Single cell suspensions of spleens were prepared by mechanical disruption. Surface staining for flow cytometry was performed using standard techniques (Johnston et al., 2009 (link)) and the following clones: CD4 (RM4-5), CD45.1 (A20), CD44 (IM7) and CD62L (MEL-14) (eBiosciences); CD8 (53-6.7) and B220 (RA3-6B2) (BD Biosciences); as well as CD8 (53-6.7), CD127 (A7R34), KLRG-1 (2F1), CD90.1 (OX-7), SLAM (TC15-12F12.2) (BioLegend). CXCR5 staining was performed as described (Choi et al., 2013 ). Intracellular staining after surface stains was performed using the “Foxp3 staining buffer” set (eBiosciences), using anti-Bcl6 monoclonal antibody (K112-91, BD Biosciences), anti-Tbet (4B10) or anti-Granzyme B (GB11) (Biolegend).
Chen R., Bélanger S., Frederick M.A., Li B., Johnston R.J., Xiao N., Liu Y.C., Sharma S., Peters B., Rao A., Crotty S, & Pipkin M.E. (2014). In vivo RNA interference screens identify regulators of antiviral CD4+ and CD8+ T cell differentiation. Immunity, 41(2), 325-338.
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2

Murine Immune Cell Phenotyping

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Antibodies and reagents used for experiments include anti-CD11c (N418), anti-CD11b (M1/70), anti-CD8a (53–6.7), anti-B220 (RA3–6B2), anti-CD49B (DX5), anti-CD25 (PL61), Streptavidin (405232), anti-CD4 (GK1.5), anti-CD45.2 (104), anti-CD62L (Mel-14), Ly-6C (AL-21), anti-IFN-γ (XMG1.2), anti-CD3 (145–2C11), Rat IgG1 Isotype control (RTK2071), anti-CD28 (37.51), anti-CD5 (53–7.3), anti-VISTA (MIH63), anti-T-bet (4B10), (BioLegend), anti-CD69 (H1.2F3), anti-RORgt (q31–378), anti-TCR V beta 11 (RR3–15), anti-Nur77 (12.14), (BD), anti-TCR V alpha 3.2 (RR3–16), anti-Foxp3 (FJK-16 s), Fc-block (anti-CD16/32) (2.4G2), (ATCC).
Opejin A., Surnov A., Misulovin Z., Pherson M., Gross C., Iberg C.A., Fallahee I., Bourque J., Dorsett D, & Hawiger D. (2020). A Two-Step Process of Effector Programming Governs CD4+ T Cell Fate Determination Induced by Antigenic Activation in the Steady State. Cell reports, 33(8), 108424.
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3

Comprehensive Immune Cell Profiling

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The following fluorophore-conjugated antibodies were used for flow cytometry: anti-CD8 (53–6.7), anti-Thy1.1 (OX-7), anti-Vα2 (B20.1), anti-CD45 (30-F11), anti-PD-1 (29F.1A12), anti-CXCR3 (CXCR3–173), anti-IFNγ (XMG1.2), anti-IL-2 (JES6–5H4), anti-TNFα (MP6-XT22), anti-CD28 (37.51), anti-Tbet (4B10) (Biolegend); anti-CD27 (LG.7F9), anti-LAG3 (C9B7W), anti-granzyme B (Ngzb), anti-Eomes (Dan11mag) (Thermo Fisher); anti-pCD3ζ (pY142) (K25–407.69), anti-CXCR5 (2G8) (BD Biosciences).
Snook J.P., Soedel A.J., Ekiz H.A., O’Connell R.M, & Williams M.A. (2020). Inhibition of SHP-1 expands the repertoire of antitumor T cells available to respond to immune checkpoint blockade.. Cancer immunology research, 8(4), 506-517.
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4

Intracellular Cytokine and Phospho-Protein Analysis

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Intracellular cytokine staining was performed as previously described [5 ]. Cell viability was determined with fixable viability dye eF660 or eF780 from eBioscience. The following antibodies were used: anti-IFN-γ (XMG1.2), anti-IL-17a (TC11-18H10.1), anti-CD4 (RM4–5), and anti-CD4 (GK1.5) (Biolegend). Cells from OT2 TCROVA recipients were stained with anti-CD45.2 (104)(Biolegend) and anti-CD45.1 (A20) (BD).
For phospho flow cytometry, cells were fixed for 7 min in 0.4% paraformaldehyde at room temperature and stained with fixable viability dye. After at an additional 7 mins in 4% paraformaldehyde the cells were permeablized in 90% methanol for 20 mins at 4°C. Cells were then stained with antibodies to pY701-STAT1 (58D6), pY705-STAT3 (D3A7), pS9-GSK3β (D85E12), pS473-AKT (D9E), pS235/236-S6 (D57.2.2E), Ku80 and/or β catenin (L54E2)(from Cell Signaling) and anti-CD4. Other reagents used as needed were goat anti-rabbit 488 (Jackson Immunoresearch), anti-RORγ(AFKJS-9, eBioscience), anti-RORγ (B2D) (eBioscience), anti-Tbet (4B10)(Biolegend). Tyramide amplification of pS473-AKT was performed using the kit (Molecular Probes). All samples were analyzed using a BD Facs Calibur or BD LSR2 and analyzed with FlowJo software.
McGuire D.J., Rowse A.L., Li H., Peng B.J., Sestero C.M., Cashman K.S., De Sarno P, & Raman C. (2014). CD5 ENHANCES TH17 DIFFERENTIATION BY REGULATING IFN-γ RESPONSE AND RORγT LOCALIZATION. European journal of immunology, 44(4), 1137-1142.
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5

Regulatory T Cell Staining Protocol

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Cells were stained for Foxp3 flow cytometry analysis according to the staining procedure described in the Mouse Regulatory T Cell Staining Kit # 3 (eBioscience, San Diego, CA, USA). We did not stimulate the cells prior to cytokine staining since it could have caused artefacts upon Foxp3 staining37 (link).
Antibodies from eBioscience were as follows: anti-CD4 (RM4-5), anti-CD25 (PC61.5), anti-CD38 (90), anti-CD39 (24DMS1), anti-CD44 (IM7), anti-Eos (ESB7C2) and anti-Foxp3 (FJK-16s). The following antibodies were from Biolegend, San Diego, USA: anti-Bcl2 (BCL/10C4), anti-Helios (22F6), anti-IFN-γ (XMG1.2), anti-IL-2 (JES6-5H4), anti-IL-17 (TC11-18H10.1) and anti-T-bet (4B10). The anti-CD8 (53-6.7) was from BD and anti-Nrp1 (761705), anti-Ebi3 (355022) and anti-IL-12p35 (27537) were from R&D, Minneapolis, MN, USA.
The stained cells were analyzed on a FACSCalibur or BD LSRII Flow Cytometry (BD) at the core facility (BioVis) Uppsala University, Uppsala, Sweden. The data were analyzed with Diva 6.0 software (BD) or Flowlogic software (Inivai Technologies, Australia). Gating strategies were made using single stained and fluorescence minus one-stained controls. Manufacturer’s instructions (BD) were followed strictly while analyzing the data and performing the flow cytometry staining.
Note: When we refer to CD4+ in thymic glands in this paper, it means CD4+CD8− and CD8+ means CD8+CD4−.
Singh K., Kadesjö E., Lindroos J., Hjort M., Lundberg M., Espes D., Carlsson P.O., Sandler S, & Thorvaldson L. (2015). Interleukin-35 administration counteracts established murine type 1 diabetes – possible involvement of regulatory T cells. Scientific Reports, 5, 12633.
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6

Murine Immune Cell Phenotyping

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Antibodies and reagents used for experiments include anti-CD11c (N418), anti-CD11b (M1/70), anti-CD8a (53–6.7), anti-B220 (RA3–6B2), anti-CD49B (DX5), anti-CD25 (PL61), Streptavidin (405232), anti-CD4 (GK1.5), anti-CD45.2 (104), anti-CD62L (Mel-14), Ly-6C (AL-21), anti-IFN-γ (XMG1.2), anti-CD3 (145–2C11), Rat IgG1 Isotype control (RTK2071), anti-CD28 (37.51), anti-CD5 (53–7.3), anti-VISTA (MIH63), anti-T-bet (4B10), (BioLegend), anti-CD69 (H1.2F3), anti-RORgt (q31–378), anti-TCR V beta 11 (RR3–15), anti-Nur77 (12.14), (BD), anti-TCR V alpha 3.2 (RR3–16), anti-Foxp3 (FJK-16 s), Fc-block (anti-CD16/32) (2.4G2), (ATCC).
Opejin A., Surnov A., Misulovin Z., Pherson M., Gross C., Iberg C.A., Fallahee I., Bourque J., Dorsett D, & Hawiger D. (2020). A Two-Step Process of Effector Programming Governs CD4+ T Cell Fate Determination Induced by Antigenic Activation in the Steady State. Cell reports, 33(8), 108424.
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7

Multicolor Flow Cytometry of Immune Cells

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Fluorophore- or biotin-conjugated antibodies specific for mouse cell-surface antigens and cytokines were as follows: anti-B220 (RA3-6B2, BioLegend), anti-CD4 (GK1.5, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD11b (M1/70, BioLegend), anti-Gr1 (RB6-8C5, BioLegend), anti-CD25 (3C7, BioLegend), anti-Foxp3 (MF14, eBioscience), anti-TER119 (TER-119, BioLegend), anti-CD71 (R17 217.1.3, BioXCell for depletion; C2F2, BD biosciences for FACS analysis), anti-CD45 (I3/2.3, BioLegend), anti-CD90.1 (16-10A1, BioLegend), anti-Gzmb (GL1, BioLegend), anti-IFN-γ (XMG1.2, BioLegend). anti-CXCR5 (L138D7, BioLegend), anti-mouse/human CD44 (IM7, BioLegend), anti-human/mouse Bcl-6 (7D1, BioLegend), anti-T-bet (4B10, BioLegend), anti-mouse Ki-67 (16A8, BioLegend), anti-mouse TNF-α (MP6-XT22, BD). For human studies, the following fluorophore- or biotin-conjugated antibodies specific for cell surface markers or cytokines were used: anti-CD3 (UCHT, BioLegend), anti-CD8 (PRA-T8, BioLegend), anti-CD45 (2D1, BioLegend), anti-CD71 (CY1G4, BioLegend), anti-CD235a (HI264, BioLegend). ROS production was measured by labelling with 2′,7′ –dichlorofluorescin diacetate using the ROS detection kit (S0033, Beyotime).
Zhao L., He R., Long H., Guo B., Jia Q., Qin D., Liu S.Q., Wang Z., Xiang T., Zhang J., Tan Y., Huang J., Chen J., Wang F., Xiao M., Gao J., Yang X., Zeng H., Wang X., Hu C., Alexander P.B., Symonds A.L., Yu J., Wan Y., Li Q.J., Ye L, & Zhu B. (2018). Late-stage tumors induce anemia and immunosuppressive extramedullary erythroid progenitor cells. Nature medicine, 24(10), 1536-1544.
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8

Phenotypic Analysis of Thymic T Cells

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Single-cell suspensions were prepared from the thymus by manual disruption using syringe plunger. PBS57-CD1d1 tetramer was obtained from the National Institutes of Health Tetramer Core Facility. The complete list of surface antibodies used is as follows: Anti-CD5 (53-7.3), anti-CD6 (BX222), anti-Ly6c (AL-21), anti-Vβ2 (B20.6), anti-Vβ7 (TR310), anti-Vβ8.1-8.2 (MR5-2), anti-Vβ8.3 (1B3.3), anti-Egr2 (erongr2), anti-CD24 (M1/69), anti-CD69 (H1.2F3). From BD Biosciences: anti-TCRβ (H57-597), anti-CD8α (53-6.7); from BioLegend: anti-CD44 (IM7); anti-CD4 (RM4-5). Surface antibody staining was done then cells were fixed and permeabilized using the FoxP3 buffer set (eBioscience). Fixed and permeabilized cells were incubated with intracellular antibodies including anti-PLZF (Mags.21F7; eBioscience), anti-Tbet (4B10; BioLegend), and anti-Rorγt (Q31-378; BD Biosciences). Cells were analyzed on a BD LSRFortessa (BD Biosciences) and data were processed with FlowJo software (TreeStar).
Tuttle K.D., Krovi S.H., Zhang J., Bedel R., Harmacek L., Peterson L.K., Dragone L.L., Lefferts A., Halluszczak C., Riemondy K., Hesselberth J.R., Rao A., O’Connor B.P., Marrack P., Scott-Browne J, & Gapin L. (2018). TCR signal strength controls thymic differentiation of iNKT cell subsets. Nature Communications, 9, 2650.
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9

Multiparameter Flow Cytometry Analysis

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For flow cytometry, cells were stained with following antibodies: anti-CD3ε (145-2C11), anti-CD8 (53-6.7), anti-CD4 (GK1.5), anti-CD24 (M1/69), anti-CD45.1 (A20), anti-CD45.2 (104), anti-B220 (RA3-6B2), anti-NKG2D (CX5), anti-IL-2Rβ (TM-β1), anti-CD94 (18d3), anti-IFN-γ (XMG1.2), anti-IL-4 (11B11), and anti-T-bet (4B10) were purchased from BioLegend. Anti-NK1.1 (PK136), anti-TCRβ (H57-597), anti-CD44 (IM7), anti-IgG1 (A85-1), anti-CD1d (1B1), and anti-CD90.2 (53-2.1) were obtained from BD Biosciences. Anti-IL-17 (ebio17B7) and anti-Rorγt (AFKJS-9) were purchased from eBioscience. Anti-PLZF (D-9) was obtained from Santa Cruz Biotechnology. Anti-CXCR3 (220803) was purchased from R&D Systems. Anti-S100a6 (EPNCIR121) was obtained from Abcam. Anti-CD16/CD32 (2.4G2) was acquired from Bio-Xcell (Malaysia).
Beyaz S., Kim J.H., Pinello L., Xifaras M.E., Hu Y., Huang J., Kerenyi M.A., Das P.P., Barnitz R.A., Herault A., Dogum R., Haining W.N., Yilmaz Ö.H., Passegue E., Yuan G.C., Orkin S.H, & Winau F. (2016). The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells. Nature immunology, 18(2), 184-195.
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10

Multiparameter Flow Cytometry Analysis

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For flow cytometry, cells were stained with following antibodies: anti-CD3ε (145-2C11), anti-CD8 (53-6.7), anti-CD4 (GK1.5), anti-CD24 (M1/69), anti-CD45.1 (A20), anti-CD45.2 (104), anti-B220 (RA3-6B2), anti-NKG2D (CX5), anti-IL-2Rβ (TM-β1), anti-CD94 (18d3), anti-IFN-γ (XMG1.2), anti-IL-4 (11B11), and anti-T-bet (4B10) were purchased from BioLegend. Anti-NK1.1 (PK136), anti-TCRβ (H57-597), anti-CD44 (IM7), anti-IgG1 (A85-1), anti-CD1d (1B1), and anti-CD90.2 (53-2.1) were obtained from BD Biosciences. Anti-IL-17 (ebio17B7) and anti-Rorγt (AFKJS-9) were purchased from eBioscience. Anti-PLZF (D-9) was obtained from Santa Cruz Biotechnology. Anti-CXCR3 (220803) was purchased from R&D Systems. Anti-S100a6 (EPNCIR121) was obtained from Abcam. Anti-CD16/CD32 (2.4G2) was acquired from Bio-Xcell (Malaysia).
Beyaz S., Kim J.H., Pinello L., Xifaras M.E., Hu Y., Huang J., Kerenyi M.A., Das P.P., Barnitz R.A., Herault A., Dogum R., Haining W.N., Yilmaz Ö.H., Passegue E., Yuan G.C., Orkin S.H, & Winau F. (2016). The histone demethylase UTX regulates the lineage-specific epigenetic program of invariant natural killer T cells. Nature immunology, 18(2), 184-195.
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