Tcrβ pacific blue

Manufactured by BioLegend

Sourced in United States

TCRβ-Pacific Blue is a lab equipment product that is used for the detection and analysis of T cell receptor beta chain expression on cells. It utilizes the Pacific Blue fluorescent dye to label the TCRβ molecule for flow cytometric analysis.

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5 protocols using tcrβ pacific blue

Multi-Marker Immune Cell Analysis

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The following antibodies were used for staining: CD4 PerCp-Cy5.5, TCRβ PerCp-Cy5.5, and CD62L APC (TONBO); CD44 FITC, PD-1 (29F.1A12) PE-Cy7, CD69 PE-Cy7, CD5 PerCp-Cy5.5, CD8α Pacific Blue, and TCRβ Pacific Blue (BioLegend); CD44 PE-Cy7, CD8α (APC-eFluor 780), CD4 Qdot606, and Zap70 FITC (Life Technology). In vivo anti-PD (J43) antibody and control IgG were purchased from BioXcell. For intracellular flow cytometry, antibodies against phosphorylated ERK^T202/Y204 (197G2), AKT^Thr308 (C31E5E), and ribosomal protein S6^Ser235/236 (D57.2.2E) were purchased from Cell Signaling Technology.

Hsu L.Y., Cheng D.A., Chen Y., Liang H.E, & Weiss A. (2017). Destabilizing the autoinhibitory conformation of Zap70 induces up-regulation of inhibitory receptors and T cell unresponsiveness. The Journal of Experimental Medicine, 214(3), 833-849.

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Comprehensive Immune Cell Profiling

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The following antibodies were used for staining: CD4 BUV395, CD8α BUV737 (BD); CD5 PerCp-Cy5.5, CD25 PE or APC, CD44 FITC or Pacific Blue, CD45.2 PE, CD62L APC, CD69 APC, TCRβ Pacific Blue, Vα2 PE or Pacific Blue, CXCR5-biotin, PD-1 FITC, SLAM PE-Cy7, CD95 PE-Cy7, GL-7 FITC (Biolegend); B220 efluore780, CD8α efluore780, ZAP70 Alexa488, Strepavidin-A647 (Life Technology); GP66 tetramer PE or APC (NIH Tetramer Core Facility). The following antibodies were used for immunoblot analysis: ZAP70 (clone 1E7.2) (36 (link)); ZAP70-pTyr³¹⁹ (clone 65E4), LAT-pTyr¹⁹¹, Erk1/2, Erk1/2-pThr²⁰²/pTyr²⁰⁴, and PLCγ-pTyr¹⁸³, all from Cell Signaling Technology; anti-phosphotyrosine (4G10), PLCγ (Sigma-Aldrich), LAT (clone FL-233, Santa Cruz Biotechnology), FLAG (clone M2, Sigma-Aldrich), and GAPDH (clone 6C5, Santa Cruz Biotechnology).

Shen L., Matloubian M., Kadlecek T.A, & Weiss A. (2021). A disease-associated mutation that weakens ZAP70 autoinhibition enhances responses to weak and self ligands. Science signaling, 14(668), eabc4479.

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Dissecting Murine Lymphoid Cell Subsets

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Lymphoid cells from bone marrow, thymus and spleen were isolated from 8-week old mice. To reveal T cell progenitor subsets in the thymus, single cell suspensions were stained with conjugated antibodies, specific for CD3-FITC (Biolegend), CD4-APC, CD8a-PerCp-Cy5.5, CD25-PE, CD44-APCCy7 (Biolegend), TCRβ-Pacific Blue (Biolegend). To analyse lymphoid subsets in the spleen, single cell suspensions were stained with CD3-FITC, CD4-APC, CD8a-PerCp-Cy5.5, CD19-APCH7, CD45R(B220)-PacificBlue, IgD-PE (eBioscience), IgM-PECy7 (eBioscience). To define the B cell progenitor subsets, suspensions from the bone marrow were stained with IgD-FITC, CD25-PE, IgM-PECy7 (eBioscience), CD45R (B220)-PacificBlue, CD117 (cKit)-APC (eBioscience), CD19-APCH7. Dead cells were excluded from the analysis by propidium iodide staining. Antibodies were purchased from BD Pharmingen unless mentioned otherwise. Samples were measured on a FACS Fortessa^® and analysed using FlowJo^® software (Version: 10.0.8r1).

Pilzecker B., Buoninfante O.A., Pritchard C., Blomberg O.S., Huijbers I.J., van den Berk P.C, & Jacobs H. (2016). PrimPol prevents APOBEC/AID family mediated DNA mutagenesis. Nucleic Acids Research, 44(10), 4734-4744.

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Characterization of Airway Immune Cells

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Immune cells were harvested from the airways via bronchoalveolar lavage (BAL). Briefly, mice were cannulated via a small tracheal incision and the lungs were flushed with 1 mL of sterile PBS. The collected BAL fluids were centrifuged at 1,500 rpm for 5 min at 4°C, and total cells were prepared for flow cytometry staining to determine the number and types of cells. Fc-blocked (1 μg/mL; eBiosciences) BALF cells were stained with anti-mouse SiglecF-PE (0.3 μg/mL; BD Pharmingen), CD11c-APC (0.3 μg/mL; eBiosciences), CD11b-PerCP (0.3 μg/mL; BioLegend), CD19-PECy5 (0.8 μg/mL; eBiosciences), Ly6G-PECy7 (0.8 μg/mL; BioLegend), Ly6C-APC-Cy7 (0.8 μg/mL; BD Pharmingen), and TCRβ-Pacific Blue (0.3 μg/mL; BioLegend). All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed by using FlowJo software (Tree Star Inc.).

dos-Santos J.S., Firmino-Cruz L., da Fonseca-Martins A.M., Oliveira-Maciel D., Perez G.G., Roncaglia-Pereira V.A., Dumard C.H., Guedes-da-Silva F.H., Santos A.C., Leandro M.D., Ferreira J.R., Guimarães-Pinto K., Conde L., Rodrigues D.A., Silva M.V., Alvim R.G., Lima T.M., Marsili F.F., Abreu D.P., Ferreira Jr. O.C., Mohana Borges R.D., Tanuri A., Souza T.M., Rossi-Bergmann B., Vale A.M., Silva J.L., de Oliveira A.C., Filardy A.D., Gomes A.M, & de Matos Guedes H.L. (2022). Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum. Frontiers in Immunology, 13, 884760.

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Phospho-Erk1/2 Analysis in Thymocytes

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2 × 10⁶ thymocytes were left for 10 min at 37 °C and stained with CD4-PE-Cy5, CD8-APC-Cy7, CD5-FITC, CD69-PE, and TCRβ-Pacific Blue (BioLegend, San Diego, CA, USA). Subsequently, cells were fixed and permeabilized for 10 min with 3.7% PFA (ChemCruz Biochemicals, Santa Cruz, CA, USA) + 0.2% saponin (Sigma Aldrich, Saint Louis, MO, USA). Subsequently, samples were washed and incubated with a phospho-specific Erk1/2 antibody (Cell Signaling, Danvers, MA, USA) in PBS containing 0.1% saponin, 0.1% sodium azide, and 0.2% BSA. Cells were washed and stained with APC-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Samples were analyzed on a FACS Fortessa I (BD Biosciences, Franklin Lakes, NJ, USA).

Kästle M., Merten C., Hartig R., Plaza-Sirvent C., Schmitz I., Bommhardt U., Schraven B, & Simeoni L. (2022). Type of PaperY192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection. International Journal of Molecular Sciences, 23(13), 7271.

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