Anti tcrβ

Manufactured by Thermo Fisher Scientific

The Anti-TCRβ is a laboratory equipment product used for the detection and analysis of T-cell receptor beta (TCR-β) chains. It is a tool for researchers to study T-cell biology and immune responses.

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Lab products found in correlation

Close spelling variants of this product

Anti-TCRβ-PE FITC-conjugated anti-mouse TCRβ (clone H57-597, Anti-mouse TCRβ (H57–597; Anti-TCRβ (H57-597, Anti-mouse TCRβ Anti-TCRβ-APC-eFluor-780 Anti-TCRβ (clone H57-597, APC‐conjugated anti‐mouse TCRβ

8 protocols using anti tcrβ

Immunofluorescence Imaging of Aortic Cells

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Harvested aortic tissues were fixed in 4% paraformaldehyde for 24 hours
and cryopreserved in 30% sucrose for 24 hours. Tissues were embedded in OCT
compound (Fisher HealthCare) and stored at −80°C. Tissues were
sectioned at 5μm thickness with a cryostat. The sections were incubated
with monoclonal anti-CD68 (1:100; Bio-Rad, Cat# MCA1957), anti-CD3 (1:100;
Invitrogen, Cat# ma5–14524) or anti-TCRβ (1:100; eBioscience, Cat#
16-5961-82) overnight at 4°C and corresponding Biotin-SP-conjugated
secondary antibodies (1:200; Jackson ImmunoResearch Laboratories) for 40 minutes
and Alexa Fluor 594 streptavidin (1:200; Molecular Probes, Cat# S11227) for 30
minutes at room temperature. Sections were mounted with Vectashield mounting
media with Dapi (Vector Laboratories, Cat# H-1200). 60× images were
captured with Keyence BZ-X710fluorescent microscope. 40× tile images were
also captured and XY-stitching was performed to recreate the full image of the
aortic tissue for quantification. Cell counting was performed with ImageJ with
the Cell Counter plugin and normalized to the measurement of outer medial
circumference.

Lu S., Strand K.A., Mutryn M.F., Tucker R.M., Jolly A.J., Furgeson S.B., Moulton K.S., Nemenoff R.A, & Weiser-Evans M.C. (2019). PTEN protects against Angiotensin II-induced pathological vascular fibrosis and remodeling. Arteriosclerosis, thrombosis, and vascular biology, 40(2), 394-403.

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Isolation and Analysis of Immune Cells

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Tongues were harvested and digested with Collagenase IV (0.7 mg/ml) in Hanks’ balanced salt solution for 30–45 mins at 37°C. Cells were separated by Percoll gradient centrifugation. Abs were from the following sources: anti-CD45 and anti-CD11b (BioLegend), anti-CD4 and anti-F4/80 (Invitrogen), anti-TCRβ, anti-CD8, and anti-CD19 (eBioscience), and anti-Ly6G and anti-Ly6C (BD Biosciences). Dead cells were excluded using Ghost Dye (eBioscience). Data were acquired with an LSRFortessa and analyzed using FlowJo software (TreeStar).
Collected single-cell suspensions from LN were filtered and dead cells were excluded using Ghost Dye (eBioscience). LN were cultured in complete medium (RPMI media containing 10% FCS, supplemented with L-glutamine and antibiotics) with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Golgiplug (BD Biosciences) for 4 h, followed by staining with Ghost Dye, CD4 and IL-17 (Biolegend).

Taylor T.C., Li Y., Li D.D., Majumder S., McGeachy M., Biswas P.S., Gingras S, & Gaffen S.L. (2022). Arid5a mediates an IL-17-dependent pathway that drives autoimmunity but not antifungal host defense. Journal of immunology (Baltimore, Md. : 1950).

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Multiparameter Flow Cytometry of Immune Cell Subsets

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Eight-color flow cytometry was conducted using total mononuclear cell preparations and was run on the MACSQuant Analyzer (Miltenyi, Auburn, CA). Antibodies purchased from eBioscience are as follows: anti-CD11b (M1/70); anti-CD18 (M18/2); anti-Ly-6G (RB6-8C5); anti-CD22 (2D6); anti-CD69 (H1.2F3); anti-MHC II (M5/114.15.2); anti-TCRβ (H57-597); anti-CD44 (IM7); anti-CD14 (Sa2-8); anti-CD45 (30-F11); anti-CD62L (MEL-14); anti-CD209 (5H10); anti-CD20 (2H7); anti-IFNγ (XMG1.2); anti-TNF-α (MP6-XT22); anti-FoxP3 (FJK-16s); and anti-IL-6 (MP5-20F3). Anti-CD8a-VioBlue was purchased from Miltenyi Biotech. Antibodies made in-house include: anti-CD4 (GK1.5); anti-CD40 (1C10); anti-CD25 (PC61.5.3); anti-CD5 (53-7.313); and anti-CD3 (145-2C11). Single cell suspensions were incubated on ice with appropriate antibodies for 30 min. Cells were washed with running buffer 3 times fixed in 1% paraformaldehyde/PBS, and then suspended in running buffer for flow cytometry. Results were analyzed with FlowJo software.

Deng G., Carter J., Traystman R.J., Wagner D.H, & Herson P.S. (2014). Pro-inflammatory T-Lymphocytes rapidly infiltrate into the brain and contribute to neuronal injury following cardiac arrest and cardiopulmonary resuscitation. Journal of neuroimmunology, 274(0), 132-140.

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Lung and Tumor Immune Cell Isolation

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Lymphocytes were isolated from lung and tumor tissue by digestion with collagenase A (1 mg/ml; Roche) and DNase I (0.5 µg/ml; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% l-glutamine, 1% penicillin-streptomycin, and 10 mM Hepes) for 30 min at 37°C. Cells were filtered through 100-µm cell strainers, washed in isolation buffer, and stained in PBS supplemented with 0.25% BSA, 2 mM EDTA, and 0.1% sodium azide. Antibodies used included anti-CD45, anti-Foxp3, anti–IL-18Rα, anti-ST2, anti-CD62L, anti-CD103, anti–PD-1, anti-GITR, anti–CTLA-4, anti-KLRG1, anti-Ki67, anti-CD5, anti-NK1.1, anti-CD45R (B220), anti–IL-17, anti–IL-4, anti-IFNγ, and anti–Ly-6C (eBioscience); anti-TCRβ, anti-CD3, anti-CD4, anti-CD8, anti-CD127, anti-CD11B, anti-MHCII, and anti-Gr1 (BioLegend); anti-CD44 and anti-CD25 (Tonbo); anti–IL-5 and anti-TNFα (BD PharMingen); and anti-AREG (R&D Systems). For exclusion of dead cells, samples were first stained with Ghost Dye (Tonbo) cell viability reagent. Intracellular staining for cytokines, Foxp3, AREG, Ki-67, and CTLA-4 was performed using the Foxp3/transcription factor staining buffer set (eBioscience) as per manufacturer’s protocol. The H2-K^b OVA_257–264 tetramer was obtained from the NIH Tetramer Core Facility.

Green J.A., Arpaia N., Schizas M., Dobrin A, & Rudensky A.Y. (2017). A nonimmune function of T cells in promoting lung tumor progression. The Journal of Experimental Medicine, 214(12), 3565-3575.

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Activating and Profiling Immune Cells

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pI:C, PMA, ionomycin and bredfeldin A were purchased from Sigma-Aldrich. Anti-CD4, anti-CD3, anti-TCRβ, anti-CD19, anti-CD25, anti-CD69, anti-DX5, anti-CD11b, anti-CD11c, anti-NK1.1, anti-IFN-γ and isotype-matched control antibodies were purchased from eBioscience. Blocking antibodies against CD1d, IL-12 and IL-18 were also purchased from eBioscience.

Zhu K., Yang J., Luo K., Yang C., Zhang N., Xu R., Chen J., Jin M., Xu B., Guo N., Wang J., Chen Z., Cui Y., Zhao H., Wang Y., Deng C., Bai L., Ge B., Qin C.F., Shen H., Yang C.F, & Leng Q. (2015). TLR3 Signaling in Macrophages Is Indispensable for the Protective Immunity of Invariant Natural Killer T Cells against Enterovirus 71 Infection. PLoS Pathogens, 11(1), e1004613.

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Multiparametric Flow Cytometry of Immune Cells

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Antibody staining was performed on tissue single-cell suspensions for 20-30 minutes at 4°C in Hanks' Balanced Salt Solution staining buffer and then washed twice with cold PBS. The sole exception was CD127, where staining was performed at room temperature for an hour. To measure cytokine production, cells were stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA), ionomycin (Iono), and Brefeldin A (BFA) for 2 hours and 30 minutes. For intracellular staining, cells were fixed for 30 minutes with the eBioscience Fixation/Permeabilization kit as described by the manufacturer and stained intracellularly for at least 1 hour with anti-CD4, anti-CD8, anti-CD90.2, anti-TCRβ, anti-IFN-γ anti-IL-17A and anti-IL17F. Antibodies for flow cytometry were purchased from BD Biosciences, BioLegend, or eBiosciences. Antibodies used were conjugated to FITC, AF488, PE, PerCP-Cy5.5, PCP-eFluor 710, PeCy7, Alexa Fluor 780, Pacific Blue, BV605, BV650, BV 510, BV785, eFluor 450, APC, Alexa Flour 647, PE Texas Red, PE-CF594. DAPI or Live/Dead fixable stain (Life Technologies) was used to exclude dead cells in all experiments. Flow cytometric data was acquired on an LSR II or LSR Fortessa (BD Biosciences) and analyzed using the FlowJo software (Tree Star, Version 9).

Huang X., Hurabielle C., Drummond R.A., Bouladoux N., Desai J.V., Sim C.K., Belkaid Y., Lionakis M.S, & Segre J.A. (2020). Murine model of colonization with fungal pathogen Candida auris to explore skin tropism, host risk factors and therapeutic strategies. Cell host & microbe, 29(2), 210-221.e6.

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Multiparametric Flow Cytometric Analysis

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Fluorescent dye–conjugated antibodies were purchased from BD Biosciences, Biolegend or Ebioscience (Thermofisher). The following clones were used: anti-CD45.1, A20; anti-Foxp3, FJK-16s; anti-CD4, RM4–5; anti-CD45.2, 104; anti-CD8α, 53–6.7; anti-CD8β, YTS 156.7.7; anti-CD44, IM7; anti-CD45, 30-F11; anti-CD62L, MEL-14; G8.8; anti-TCRβ, H57–597; anti-TCRγδ, eBioG23; anti-CD25, PC61.5;. Live/dead fixable dye Aqua (ThermoFisher Scientific) was used according to manufacturer’s instructions. Intracellular staining of Foxp3 was conducted using Foxp3 Mouse Regulatory T Cell Staining Kit (eBioscience, USA). Flow cytometry data was acquired on a LSR-II flow cytometer (Becton Dickinson, USA) and analyzed using FlowJo software package (Tri-Star, USA). Anti-ThPOK ChIP antibody was kindly provided by T. Egawa (Wash. U.).

London M., Bilate A.M., Castro T.B., Sujino T, & Mucida D. (2021). Stepwise chromatin and transcriptional acquisition of an intraepithelial lymphocyte program. Nature immunology, 22(4), 449-459.

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Intestinal Epithelial Cell Phenotyping

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Colon cells were stained with Fc Block (Clone 2.4G2) followed by staining with fluorescently labelled antibodies in IEC buffer (PBS with 5% FBS and 2 mM EDTA to reduce cell clumping) for IECs or 2% FBS in PBS for T cells on ice in 1.5 ml microcentrifuge tubes. For intracellular staining, cells were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Bioscience). Samples were acquired on an Attune NxT flow cytometer (Life Technologies) and analysed with FlowJo software. Cells were sorted on either a BD ARIA II or S6 Enceladus (BD Biosciences). The following antibodies/reagents were used: anti-CD45 (30-F11; Biolegend), anti-EPCAM1 (G8.8; ThermoFisher), anti-FABP2 (polyclonal; R&D/Fisher), anti-goat IgG (ThermoFisher), anti-LY6G (1A8; BioLegend), anti-MHCII (M5/114.15.2; Fisher), Live/Dead Fixable Near-IR dead cell dye (ThermoFisher), anti-CD4 (RM4-5; BioLegend), anti-TCRβ (Η57−597; ThermoFisher), anti-CD44 (IM7; BioLegend), anti-CD45.1 (A20; ThermoFisher), anti-CD45.2 (104; BD Biosciences), anti-IL-17A (TC11-18H10; BD Biosciences), anti-IFNγ (XMG1.2; ThermoFisher), and anti-IL-22 (1H8PWSR; ThermoFisher).

Zindl C.L., Wilson C.G., Chadha A.S., Duck L.W., Cai B., Harbour S.N., Nagaoka-Kamata Y., Hatton R.D., Gao M., Figge D.A, & Weaver C.T. (2024). Distal colonocytes targeted by C. rodentium recruit T-cell help for barrier defence. Nature, 629(8012), 669-678.

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