Anti tcrβ h57 597

Manufactured by Thermo Fisher Scientific

The Anti-TCRβ (H57-597) is a monoclonal antibody that recognizes the T cell receptor beta chain (TCRβ) on T cells. It is commonly used for the identification and characterization of T cell populations in flow cytometry applications.

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TCRβ (H57-597) (20 citations) H57-597 (10 citations) Anti-TCRβ (8 citations) Anti-mouse TCRβ (H57–597; (4 citations) FITC-conjugated anti-mouse TCRβ (clone H57-597, (2 citations) Anti-TCRβ (clone H57-597, (2 citations)

8 protocols using anti tcrβ h57 597

Visualizing LSEC-T Cell Interactions

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LSEC were grown on collagen-coated coverslips and loaded with 0,1 mg/ml OVA (for coculture with OT-1 T cells) or left untreated (for coculture with DesTCR T cells) for 1–2 hours. To ensure that the start of LSEC/T cell interaction was synchronized naïve T cells were centrifuged onto the LSEC for 1′ at 1000 rpm. For TIRF microscopy cells were fixed after the indicated time-points in 4% paraformaldehyde, blocked with Tris-Buffered Saline containing 1% BSA/1% donkey serum (Jackson Immunoreasearch) and stained with anti-TCRβ (H57-597, eBioscience), Alexafluor-488 Goat-anti-Hamster IgG (H+L, Molecular Probes) as secondary antibodies or anti-CD11a (I21/7, Southern Biotech) antibodies, Alexafluor-488 Goat-anti-Rat IgG (H+L, Molecular Probes) as secondary antibodies. After washing coverslips were mounted in ProlongGold (Invitrogen), supplemented with 50 µg/mL DABCO anti-fade reagent (Sigma-Aldrich), and analyzed. For confocal microscopy cells were incubated with avidin/biotin blocking agent (Invitrogen) and stained with biotinylated anti-TCRβ and unlabeled anti-CD11a antibodies and Cy5-labeled streptavidin and Cy3-labeled anti-Rat-IgG (Jackson Immunoresearch).

Kaczmarek J., Homsi Y., van Üüm J., Metzger C., Knolle P.A., Kolanus W., Lang T, & Diehl L. (2014). Liver Sinusoidal Endothelial Cell-Mediated CD8 T Cell Priming Depends on Co-Inhibitory Signal Integration over Time. PLoS ONE, 9(6), e99574.

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Comprehensive T Cell Immunophenotyping

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For the flow cytometry analysis, anti-CD4 (GK1.5), anti-CD8α (53–6.7), anti-CD44 (IM7), anti-CD127 (A7R34), anti-CD69 (FN50), anti-Bcl-2 (BCL/10C4) were purchased from Biolegend. Anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CD5 (53–7.3), anti-CD45.1 (A20), anti-CD45.2 (104), mouse lgG1 κ isotype control (556027), anti-CD25 (PC61), Rat lgG2a isotype control (eBRG1) and purified anti-human-CD3 (OKT3) were purchased from BD Bioscience. Anti-Foxp3 (FJK-16s), anti-TCRβ (H57–597) and anti-CD3 (UCHT1) were purchased from eBioscience. Fluo-4,AM (F14217) was purchased from Invitrogen. Streptavidin (NEBN7021S) was purchased from Sigma.
For western analysis, anti-α-tubulin (sc-53029) and anti-Arp3 (sc15390) antibodies were purchased from Santa Cruz Biotechnology. Anti-Arpc2 (ab96779) was purchased from Abcam.
For immunofluorescence analysis, anti-Mouse TCR β-chain-488 (H57-597) and anti-Mouse CD3-488 (17A2) were purchased from Biolegend. Anti-CD3ζ (6B10.2) was purchased from Santa Cruz. Anti-EEA1 (C45B10), anti-Rab5 (C8B1) and anti-Rab11 (D4F5) were purchased from Cell Signaling Technology. Anti-LAMP1 (ab24170) was purchased from Abcam.
Cytochalasin D (ab143484) was purchased from Abcam. Staphylococcal enterotoxin E (ET404) was purchased from Toxin Technology. CELLTRACKER(TM) BLUE CMAC (C2110) was purchased from Invitrogen.

Zhang Y., Shen H., Liu H., Feng H., Liu Y., Zhu X, & Liu X. (2017). Arp2/3 complex controls T cell homeostasis by maintaining surface TCR levels via regulating TCR+ endosome trafficking. Scientific Reports, 7, 8952.

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Comprehensive Multiparametric Flow Cytometry

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The following monoclonal antibodies (mAbs) were purchased from BD Bioscience, BioLegend, or e-Bioscience: anti-CD4 (GK1.5), anti-CD8 (53.67), anti-CD16/32 (93), anti-CD19 (6D5), anti-CD24 (M1/19), anti-CD27(LG.3A10), anti-TCRβ (H57-597), anti-CD43 (S7), anti-CD44 (1M7), anti-CD11a (M17/4), anti-CD69 (H1.2F3), anti-NKG2D (CX5), anti-NK1.1 (PK136), anti-CD103 (2E7), anti-CCR6 (29-2L17), anti-CD122 (TM-β1), anti-CD45.1 (A20), anti-CD45.2 (104), anti-EGR2 (erongr2), anti-T-bet (eBio4B10), anti-Gata3 (TWAJ), anti-Rorγ (B2D), anti-PLZF (9E12), anti-Eomes (Dan11mag), anti-IFN-γ (XMG1.2), anti-IL-4 (11B11), anti-IL-17 (TC11-18H10), anti-IL-17RB(9B10) and CD1d-dimers. CD1d-tetramers were purchased from MBL. Fixable Aqua Dead Cell stain kit (Invitrogen) was used to eliminate dead cells. Intracellular staining for transcription factors was performed using the eBioscience Foxp3 Staining Buffer Kit. Intracellular staining for cytokines was performed using the BD Cytofix/Cytoperm™ Kit. For analysis, a FACSCalibur, Canto II or LSRFortessa X-20 instrument and the CELLQuest or FACSDiva (BD Biosciences) or FlowJo software (v10.3B2) packages were used.

Shimizu K., Sato Y., Kawamura M., Nakazato H., Watanabe T., Ohara O, & Fujii S.I. (2019). Eomes transcription factor is required for the development and differentiation of invariant NKT cells. Communications Biology, 2, 150.

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Immunofluorescent Staining of Ear Pinnae

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Ear pinnae were split with forceps, fixed in 1% paraformaldehyde (Electron Microscopy Sciences) overnight at 4°C, and blocked in PBS containing 1% BSA and 0.25% Triton X-100 for 2 h at room temperature. Tissues were first stained with anti-CD4 (RM4–5, eBioscience), anti-CD8α (53–6.7, eBioscience), anti-CD49f (GoH3, eBioscience), anti-GFP (A21311, Life Technologies), and anti-TCRβ (H57–597, eBioscience) antibodies overnight at 4°C, washed three times with PBS, and then stained with DAPI (Sigma-Aldrich) overnight before being mounted with ProLong Gold (Invitrogen). Ear pinnae images were captured on a Leica TCS SP8 confocal microscope equipped with HyD and PMT detectors and a 40X oil objective (HC PL APO 40X/1.3 oil). Images were analyzed using Imaris (Bitplane).

Constantinides M.G., Link V.M., Tamoutounour S., Wong A.C., Perez-Chaparro P.J., Han S.J., Chen Y.E., Li K., Farhat S., Weckel A., Krishnamurthy S.R., Vujkovic-Cvijin I., Linehan J.L., Bouladoux N., Merrill E.D., Roy S., Cua D.J., Adams E.J., Bhandoola A., Scharschmidt T.C., Aubé J., Fischbach M.A, & Belkaid Y. (2019). MAIT cells are imprinted by the microbiota in early life and promote tissue repair. Science (New York, N.Y.), 366(6464), eaax6624.

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Cytokine Analysis of CD4+ T Cells

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Single cell suspension of PECs and LNs were stained with anti-CD45 (30-F11: eBioscience; San Diego, CA), anti-CD4 (RM4–5: Biolegend; San Diego, CA), and anti-TCRβ (H57–597: eBioscience) antibodies. Subsequently, the cells were stimulated with cell stimulation cocktail (eBioscience) and monensin (eBioscience) for 5 hours. After stimulation, the cells were stained with Ghost Dye UV450 (TONBO) to identify dead cells. After fixation and permeabilization by IC Fixation Buffer (eBioscience), intracellular cytokines were stained by anti-IFN-γ (XMG1.2: Biolegend), and anti-IL-10 (JES5–16E3: eBioscience) antibodies. Cytokine⁺ CD4⁺ T cells numbers were analyzed by flow cytometry.

Kobiyama K., Vassallo M., Mitzi J., Winkels H., Pei H., Kimura T., Miller J., Wolf D, & Ley K. (2018). A clinically applicable adjuvant for an atherosclerosis vaccine. European journal of immunology, 48(9), 1580-1587.

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Multiparametric Flow Cytometry Analysis

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For analysis of surface markers, cells were stained in PBS/1% FBS with anti-CD45 (30-F11), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-F4/80 (BM8), anti-CD11c (N418), anti-MHC-II (M5/114.15.2), anti-γδTCR (eBioGL3), anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8α (53-6.7), and anti-TCRβ (H57-597) (all from eBioscience). For intracellular staining with anti-IL-17 (eBio17B7) and anti-interferon (IFN)-γ (XMG 1.2) (all from eBioscience), cells were stimulated with PMA and ionomycin in the presence of protein transport inhibitor for 5 h before being stained according to the manufacturer’s instructions (BD Biosciences). Anti-Foxp3 (FJK-165, eBioscience) staining was done according to the manufacturer’s instructions (eBioscience). Flow cytometry data were acquired on BD FACSCannto™ II or BD LSRFortessa™ X-20 and analyzed with FlowJo software (Treestar). KCs were sorted with a BD FACSAria™ III Sorter.

Zhao W., Xiao S., Li H., Zheng T., Huang J., Hu R., Zhang B., Liu X, & Huang G. (2018). MAPK Phosphatase-1 Deficiency Exacerbates the Severity of Imiquimod-Induced Psoriasiform Skin Disease. Frontiers in Immunology, 9, 569.

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Multiparameter Flow Cytometry Analysis

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Cells were washed with PBS containing 0.5% BSA and incubated for 30 minutes on ice with the following antibodies: anti-CD4 (RM-4.5; eBioscience), anti-CD8 (53.6.7; BD), anti-CD44 (IM.7; BD), anti-CD25 (PC61; BD), anti-CD62L (MEL14; Biolegend), anti-TCRβ (H57-597; eBioscience), anti-TCRγδ (BD), anti-CD69 (H1.2F3; eBioscience), anti-CD11b (M1/70; BD), anti-Gr1 (RB6-8C5; eBioscience) and anti-HSA (M1/69, eBioscience). Cells were then washed in PBS with 0.5% BSA and data was collected using a Fortessa cytometer (BD Bioscience) and analyzed using FlowJo software (Treestar, Ashland, OR).
In the case of intracellular staining, cells were fixed and permeabilized using the Foxp3 buffer staining kit (eBioscience) according to the manufacturer’s instructions prior to staining for intracellular Foxp3 expression using an anti-Foxp3 antibody (FJK-16s, BD), for 30 minutes.
For Annexin V staining, cells were washed with PBS and stained with BD Pharmingen Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.
The detection of BrdU was performed using BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions.

Prochazkova J., Sakaguchi S., Owusu M., Mazouzi A., Wiedner M., Velimezi G., Moder M., Turchinovich G., Hladik A., Gurnhofer E., Hayday A., Behrens A., Knapp S., Kenner L., Ellmeier W, & Loizou J.I. (2015). DNA Repair Cofactors ATMIN and NBS1 Are Required to Suppress T Cell Activation. PLoS Genetics, 11(11), e1005645.

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Analyzing T Cell Activation by TCR-CD28 Co-Stimulation

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Total or sorted DP thymocytes were cultured with 5 μg ml⁻¹ purified anti-CD28 (37.51, eBioscience) in a 96-well plate that was coated with 10 μg ml⁻¹ purified anti-TCRβ (H57-597, eBioscience) for the indicated times for apoptosis and quantitative RT-PCR analysis.

Wang J., He N., Zhang N., Quan D., Zhang S., Zhang C., Yu R.T., Atkins A.R., Zhu R., Yang C., Cui Y., Liddle C., Downes M., Xiao H., Zheng Y., Auwerx J., Evans R.M, & Leng Q. (2017). NCoR1 restrains thymic negative selection by repressing Bim expression to spare thymocytes undergoing positive selection. Nature Communications, 8, 959.

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