T bet 4b10

Manufactured by BioLegend

Sourced in United States

T-bet (4B10) is a mouse monoclonal antibody that recognizes the transcription factor T-bet. T-bet is a key regulator of Th1 cell differentiation and plays a critical role in the development and function of Th1 cells.

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Lab products found in correlation

Ghost dye red 780, by Cytek Biosciences (3 mentions) Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (3 mentions) Cd19 6d5, by BioLegend (3 mentions) Cd44 im7, by BD (3 mentions) Cd4 gk1, by BD (3 mentions) Cd8 53 6, by BD (3 mentions) Bovine serum albumin (bsa), by Avantor (2 mentions) Na2edta, by Avantor (2 mentions) Q31 378, by BD (2 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (2 mentions) Rorγt q31 378, by BD (2 mentions) Gata3 16e10a23, by BioLegend (2 mentions) Cd11b m1 70, by BD (2 mentions) Cd11c n418, by BioLegend (2 mentions) I a 1 e m5 114, by BioLegend (2 mentions) Cd11c n418, by BD (2 mentions) Cd3e 145 2c11, by BD (2 mentions) Cd45.1 a20, by BioLegend (2 mentions) F4 80 bm8, by BD (2 mentions) Fortessa, by BD (2 mentions)

17 protocols using t bet 4b10

Multiparameter Flow Cytometry of B cell Subsets

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Analysis of B cell surface proteins was determined by flow cytometry using PE-594 anti-CD19 (HIB19), Brilliant Violet (BV)605 anti-IgD (IA6–2), BV650-anti-CD27 (O323), PerCP.Cy5 anti-CD38 (HIT2), APC anti-CD21 (Bu32), Pacific Blue anti-CD11c (3.9) obtained from BioLegend, and PE anti-IL-4R (hIL4R-M57) obtained from BD Biosciences. Dead cells were excluded from analysis with APC-eFluor® 780 Organic Viability Dye (ThermoFisher Scientific).
For intracellular staining of IFN-β (clone MMHB-3, PBL Assay Science), cells were stained with ef780 viability dye, followed by fixation in 2% PFA and 70% ice-cold methanol permeabilization before staining. For intranuclear staining of T-bet (4B10, BioLegend) or IRF7 (12G9A36, BioLegend), permeabilization and fixation were carried out using the eBioscience™ Transcription Factor Staining Buffer (ThermoFisher Scientific). Cells (300,000–1 X 10⁶ per sample) were analyzed by flow cytometry. FACS data were acquired with an LSRII FACS analyzer (S/N# 30201809, BD Biosciences) and analyzed with FlowJo software v10.6.2 (Tree Star Ashland, OR).

Gao M., Liu S., Chatham W.W., Mountz J.D, & Hsu H.C. (2022). IL-4-induced quiescence of resting naïve B cells is disrupted in systemic lupus erythematosus. Journal of immunology (Baltimore, Md. : 1950), 209(8), 1513-1522.

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Immunophenotyping of T Cell Subsets

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Immunofluorescence antibodies were used as purchased. The 0.1% flow staining buffer was formulated by adding 0.1% bovine serum albumin (VWR, Radnor, PA), 2mM Na2EDTA (VWR, Radnor, PA) and 0.01% NaN3 (VWR, Radnor, PA) in DI H₂O. Flow cytometry was completed by following the manufacturer's guidance of the Attune NXT Flow cytometer (ThermoFisher Scientific, Waltham, MA, USA) in Arizona State University’s flow cytometry core. The following immunofluorescence antibodies were utilized in the flow cytometry studies: MHC-Tetramer: - I-A(q) bovine collagen II 271-285 GEPGIAGFKGEQGPK (NIH Tetramer Core Facility), CD4 (RM4-5, 566407, BD Biosciences), CD8 (53-6.7, 564983, BD Bioscience), CD25 (PC61, 552880, BD Biosciences), CD44 (IM7, 566200, BD Biosciences), Tbet (4B10, 644835, BioLegend), GATA3 (L50-823, 565449, BD Biosciences), RORyT (Q31-378, 564722, BD Biosciences), Foxp3 (FJK-16s, 48-5773-82, Invitrogen) and Ki67 (SolA15, 11-5698-82, Invitrogen).

Mangal J.L., Inamdar S., Suresh A.P., Jaggarapu M.M., Esrafili A., Ng N.D, & Acharya A.P. (2022). Short Term, Low Dose Alpha-ketoglutarate Based Polymeric Nanoparticles with Methotrexate Reverse Rheumatoid Arthritis Symptoms in Mice and Modulate T Helper Cell Responses. Biomaterials science, 10(23), 6688-6697.

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Multiparametric Analysis of Tumor-Infiltrating Lymphocytes

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Single‐cell suspensions of tumor‐infiltrating lymphocytes or TdLN cells were stained with the appropriate monoclonal antibody in phosphate‐buffered saline containing 10% rat serum. When necessary, intracellular staining was performed using the TrueNuclear Transcription Factor Buffer Set (BioLegend, San Diego, USA) according to the manufacturer's instructions. CytoFLEX (Beckman Coulter, Brea, USA) and FACSAria III (BD Biosciences, San Diego, USA) were used for analysis and cell sorting, respectively. Dead cells were excluded by the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen, Carlsbad, USA). Antibodies specific for mouse CD3 (17A2), IFN‐γ (XMG1.2), CD8α (53‐6.7), TNF‐α (MP6‐XT22), NK1.1 (PK136), CD45 (30‐F11), CD69 (H1.2F3), perforin (S16009A), TCR Vα2 (B20.1), and T‐bet (4B10) were purchased from Biolegend (San Diego, USA). Antibodies specific for mouse Eomes (Dan11mag) were purchased from Thermo Fisher (Waltham, USA). For determination of intracellular IFN‐γ expression, isolated single‐cell suspensions of tumor‐infiltrating lymphocytes or TdLN cells were stimulated with Cell Stimulation Cocktail (Thermo Fisher, Waltham, USA) for 3 h in complete RPMI 1640 medium before antibodies staining.

Bi J., Jin X., Zheng C., Huang C., Zhong C., Zheng X., Tian Z, & Sun H. (2023). Checkpoint TIPE2 Limits the Helper Functions of NK Cells in Supporting Antitumor CD8+ T Cells. Advanced Science, 10(12), 2207499.

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Tetramer-Based Immune Cell Profiling

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Spleen and lymph node cells or blood cell were harvested and stained with p:I-A^b tetramers, and in a subset of experiments with CXCR5 (L138D7; BioLegend) antibody for one hour at room temperature, then incubated with anti-PE and anti-APC magnetic beads. Samples then passed over magnetized columns (Miltenyi). Bound cells were eluted from the columns and stained for 30 minutes on ice with antibodies from eBioscience to CD3e (145-2C11), CD4 (GK1.5; BD), CD8 (53-6.7), CD90.1 (HIS51;), CD90.2 (53-2.1), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7), CD45.2 (104), B220 (RA3-6B2), and CD45.1 (A20). Cellular viability by GhostDye Red 780 (Tonbo). Stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for one hour at room temperature and stained overnight at 4 °C with antibodies to T-bet (4B10; BioLegend), FoxP3 (FJK-16s; eBioscience), RORγt (Q31-378; BD), and/ or Bcl-6 (K112-91; BD). Cells were analyzed on a Fortessa X-20 (BD) using FlowJo software v10 (TreeStar). The total number of tetramer-positive cells in the enriched fraction from each mouse was determined using AccuCheck Counting Beads (ThermoFisher).

Burrack A.L., Malhotra D., Dileepan T., Osum K.C., Swanson L.A., Fife B.T, & Jenkins M.K. (2017). Allograft rejection is associated with weak T cell responses to many different graft leukocyte-derived peptides. Journal of immunology (Baltimore, Md. : 1950), 200(2), 477-482.

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Peptide-based T-cell Activation Assay

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Ova (ova_323–339) (ISQAVHAAHAEINEAGR) and MART-1 (ELAGIGILTV) peptide were purchased from GenScript (Piscataway, NJ). Penicillin, streptomycin, glucose free RPMI-1640, IMDM were purchased from Life Technologies, Grand Island, NY. FBS was procured from BioAbChem Inc., Ladson, SC. All the recombinant cytokines except IL-2 (Shenandoah Biotechnology, Warwick, PA) and fluorochrome conjugated anti-mouse CD4 (GK1.5), CD73 (TY/11.8), CD26 (H194-112), CD44 (IM7), CD62L (MEL-14), IFN-γ (XMG1.2), IL-17a (TC11-18H10.1), IL-22 (Poly5164), IL-2 (JES6-5H4), TNF-α (MP6-XT22), CD25 (PC61) and T-bet (4B10) were purchased from BioLegend, San Diego, CA. Fluorochrome conjugated anti-mouse Vβ5.1,5.2 (MR9-4), IRF-4 (3E4), CD39 (24DMS1) and RORγt (AFKJS-9) were obtained from eBiosciences (San Deigo, CA). Anti-human Vβ12 was from Thermo Scientific (Rockford, IL). Purified anti-CD3, anti-CD28, anti-IFN-γ, anti-IL-4 were obtained from UCSF monoclonal antibody core, UCSF, CA. Anti-mouse pS6 conjugated with Alexa647 was purchased from Cell Signaling Technology (Danvers, MA). Tumor cells tested for antibody production were obtained from our collaborators as follows: 624-MEL (Dr. Michael Nishimura, Loyola University, Chicago), EL4 (Dr. Zihai Li, MUSC), B16-ova (Dr. Mark Rubinstein, MUSC).

Chatterjee S., Thyagarajan K., Kesarwani P., Song J.H., Soloshchenko M., Fu J., Bailey S., Vasu C., Kraft A., Paulos C.M., Yu X.Z, & Mehrotra S. (2014). Reduced CD73 Expression by IL-1β Programmed Th17 Cells Improves Tumor Control. Cancer research, 74(21), 6048-6059.

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Infant and Adult Murine Lung Immune Profiling

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Bronchoalveolar lavage (BAL) was collected using HBSS + 30μM EDTA; the cellular component was isolated and resuspended in HBSS for enumeration. The superior/upper right lobe (URL) was harvested and processed into a single cell suspension, as previously described^{26 (link)}. Each BAL and URL sample represents a single adult animal; infant BAL samples (2-4 infant mice) were pooled to acquire sufficient cell numbers for flow cytometry. To maintain consistency with the BAL, the same infant URL samples were pooled prior to enumeration. Cells (0.5 – 1 × 10⁶) were surface stained with antibodies against TCR_β-H57-597, CD62L-MEL-14, and CD19-6D5 (Biolegend, San Diego, CA), CD4-GK1.5, CD8a-53-6.7, CD44-IM7 (BD Biosciences, San Jose, CA). Cells were fixed and permeabilized for transcription factor staining using the BD Pharmingen Transcription Factor Buffer Set, according to manufacturer recommendations (BD Biosciences). Intracellular staining of GATA3-16E10A23 and Tbet-4B10 (Biolegend) was performed following overnight incubation in BD Fix/Perm solution. Where indicated, BAL samples were incubated with RSV F-protein MHC I pentamer (H-2kd KYKNAVTEL, ProImmune, Sarasota, FL). Samples were run on a BD LSRFortessa (BD Biosciences) managed by the University of Pittsburgh United Flow Core and analyzed using FlowJo V10 software (FLOWJO, Ashland, OR).

Eichinger K.M., Kosanovich J.L, & Empey K.M. (2017). Localization of the T-Cell Response to RSV Infection is Altered in Infant Mice. Pediatric pulmonology, 53(2), 145-153.

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Intracellular Staining of T Cell Transcription Factors

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Cells that were already stained for surface markers were fixed with 1.5% PFA (Sigma Aldrich, St. Louis, MO) for 10 min at room temperature. Intracellular staining was performed in FACS buffer (PBS, 0.5% albumin Fraction V (Roth, Karlsruhe), and 0.1% NaN₃) containing 0.1% saponin (Sigma Aldrich, St. Louis, MO). The cells were washed with the same buffer and resuspended in PBS for measurement.
For staining of transcription factors in differentiated T cells, surface marker staining was performed for 10 min on ice before the cells were fixed with True-Nuclear 1× Fix Concentration (BioLegend, San Diego, CA) for 1 h at room temperature. The T cells were permeabilized and stained for transcription factors (T-bet (4B10), BioLegend, San Diego, CA; GATA-3 (16E10A23), BioLegend, San Diego, CA; RORγt (Q21–559), BioLegend, San Diego, CA) in True-Nuclear 1× Perm Buffer (BioLegend, San Diego, CA) for 30 min at room temperature.

Orlik C., Deibel D., Küblbeck J., Balta E., Ganskih S., Habicht J., Niesler B., Schröder-Braunstein J., Schäkel K., Wabnitz G, & Samstag Y. (2019). Keratinocytes costimulate naive human T cells via CD2: a potential target to prevent the development of proinflammatory Th1 cells in the skin. Cellular and Molecular Immunology, 17(4), 380-394.

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Multiparametric Flow Cytometry Analysis

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Tetramer-enriched samples were stained for surface markers for 30 minutes on ice using antibodies to: CD4 (GK1.5, BD), CD8 (53–6.7, BD), CD90.1 (HIS51), CD90.2 (53–2.1, 30-H12), CD3e (145-2C11, BD), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD44 (IM7, BD), CD45.1 (A20, Biolegend), CD45.2 (104), B220 (RA3–6B2), and/or PD1 (J43). Cellular viability was confirmed using GhostDye Red 780 (Tonbo Biosciences). For transcription factor expression analysis, stained cells were fixed and permeablized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Cells were stained overnight at 4°C with antibodies to T-bet (4B10, Biolegend), Foxp3 (FJK-16s), RORγt (Q31-378, BD), and Bcl-6 (K112-91, BD). For thymic dendritic cell and thymic epithelial cell analyses cells, following enrichment cells were stained with antibodies to CD11c (N418), CD19 (6D5, Biolegend), I-A/I-E (M5/114.15.2, Biolegend), CD8 (53–6.7, BD), CD90.2 (53–2.1, 30-H12), CD11b (M1/70), and CD45.2 (104, BD). Antibodies were purchased from eBioscience unless otherwise indicated. Cells were analyzed by flow cytometry on a Fortessa (BD). Data were analyzed using FlowJo software (TreeStar).

Malhotra D., Linehan J.L., Dileepan T., Lee Y.J., Purtha W.E., Lu J.V., Nelson R.W., Fife B.T., Orr H.T., Anderson M.S., Hogquist K.A, & Jenkins M.K. (2016). Polyclonal CD4+ T cell tolerance is established by distinct mechanisms, according to self-peptide expression patterns. Nature immunology, 17(2), 187-195.

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MOG35-55-induced EAE Mouse Model

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Synthetic MOG35-55 was purchased from Anaspec. MBP ac1-11 was purchased from Genemed. Recombinant cytokines used in vitro include rhIL-2 at 10u/ml (Peprotech), rIL-12 at 10ng/ml (Biolegend). The following antibodies were utilized in cell culture, all were purchased from BioXcell: anti-CD3 (145-2C11), anti-CD28 (PV-1) and anti-IL-4 (clone 11B11). The following fluorophore-conjugated antibodies were used for flow cytometry. Antibodies purchased from Biolegend: CD3ε (145-2C11), CD4 (GK1.5), CD11b (M1/70), CD25 (3C7), CD44 (IM7), CD69 (H1.2F3), IFN-γ (XMG1.2), IL-17a (TC11-18H10.1) and T-bet (4B10). Antibodies purchased from BD: GM-CSF (MP1-22E9) and RORγt (Q31-378). Anti-FoxP3 (FJK-16s) was purchased from eBioscience.

Cummings M., Arumanayagam A.C., Zhao P., Kannanganat S., Stuve O., Karandikar N.J, & Eagar T.N. (2018). Presenilin1 regulates Th1 and Th17 effector responses but is not required for experimental autoimmune encephalomyelitis. PLoS ONE, 13(8), e0200752.

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Phenotypic Profiling of Immune Cells

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CD4 (clone GK1.5, Biolegend, #100453, dilution 1:200); CD8 (53-6.7, BD Biosciences, #563786, 1:200); IFNγ (XMG1.2, Biolegend, 505825, 1:100); IL-17A (TC11-18H10.1, Biolegend, #506938, 1:100); IL-22 (POLY5164, Biolegend, #516411, 1:100); RORγT (B2D, eBiosciences, #12-6981-80, 1:100); TBET (4B10, Biolegend, #644805, 1:100); AHR (4MEJJ, eBiosciences, #25-5925-80, 1:100); pSTAT3 (LUVNKLA, eBioscience, #11-903-42, 1:100); pSMAD2/3 (o72-670, BD Biosciences, #562586, 1:100)

Minns D., Smith K.J., Alessandrini V., Hardisty G., Melrose L., Jackson-Jones L., MacDonald A.S., Davidson D.J, & Gwyer Findlay E. (2021). The neutrophil antimicrobial peptide cathelicidin promotes Th17 differentiation. Nature Communications, 12, 1285.

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