Anti mouse cd3 clone 145 2c11

Manufactured by BD

Anti-mouse CD3 (clone 145-2C11) is a monoclonal antibody that recognizes the CD3 complex on mouse T cells. It is used for the identification and enumeration of T cells in mouse samples.

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Lab products found in correlation

Anti mouse cd8 clone 53 6, by BioLegend (2 mentions) Anti mouse cd19 clone 1d3, by Thermo Fisher Scientific (2 mentions) Anti δhcd4 clone rpa t4, by Thermo Fisher Scientific (2 mentions) Recombinant mouse hvem fc, by R&D Systems (2 mentions) Milliplex map mouse cd8 t cell magnetic bead panel, by Merck Group (1 mentions) Stepone system, by Thermo Fisher Scientific (1 mentions) Anti mouse cd28 clone 37.51, by BD (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Anti mouse cd8 clone 53 6, by BD (1 mentions) Model no szh illb, by Olympus (1 mentions) Tnf α elisa max standard set, by BioLegend (1 mentions) Elisa max standard set mouse ifn γ, by BioLegend (1 mentions) Il 12 elisa max standard set, by BioLegend (1 mentions) Elispot assay, by BD (1 mentions) Anti mouse cd4 clone rm4 5, by BD (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Protein assay kit, by Thermo Fisher Scientific (1 mentions) Sytox blue, by Thermo Fisher Scientific (1 mentions) Anti cd8 clone rpa t8, by BD (1 mentions) Anti human cd3 clone okt3, by Thermo Fisher Scientific (1 mentions)

7 protocols using anti mouse cd3 clone 145 2c11

Detailed Immunophenotyping of Mouse Leukocytes

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Blood (50 μl) was drawn from the tail veil every week. This was performed at least 24 hours after OXA application. Whole blood samples were processed using lysis buffer (from Hospital Pharmacy at LUMC) for 10 mins at 37°C. Cells were incubated with monoclonal antibodies for 30 min on ice.
Fluorescence-labeled antibodies including anti-mouse CD3 (clone 145-2C11, BD, The Netherlands), anti-mouse CD19(clone 1D3, Thermo Fisher Scientific, The Netherlands), anti-mosue CD4 (clone RM4-5, Thermo Fisher Scientific, The Netherlands), anti-mouse CD8 (clone 53-6.7, Biolegend, The Netherlands) and anti-ΔhCD4 (clone RPA-T4, eBioscience™, The Netherlands). Of note, the antibody for ΔhCD4 should be specifical clone that fits for the surrogated reporter in Socs1flox transgenic mouse. Samples were processed in a BD Fortessa flow cytometer and analyzed using the FlowJo software.

Luo Y., Vermeer M.H., de Gruijl F.R., Zoutman W.H., Sluijter M., van Hall T, & Tensen C.P. (2022). In vivo modelling of cutaneous T-cell lymphoma: The role of SOCS1. Frontiers in Oncology, 12, 1031052.

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Murine OT-1 BTLA Cytokine Secretion

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Murine OT-1 BTLA KO cells overexpressing BTLA WT or mutants were re-stimulated with either 10 ng/ml anti-mouse CD3 (Clone 145-2C11, BD Pharmingen™) alone or with recombinant mouse HVEM-Fc (R&D systems) plate-bound for 24 h. Supernatants were collected to quantify the secreted cytokines using a MILLIPLEX MAP Mouse CD8⁺ T Cell Magnetic Bead Panel (Millipore).

Ritthipichai K., Haymaker C.L., Martinez M., Aschenbrenner A., Yi X., Zhang M., Kale C., Vence L.M., Roszik J., Hailemichael Y., Overwijk W.W., Varadarajan N., Nurieva R., Radvanyi L.G., Hwu P, & Bernatchez C. (2017). Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(20), 6151-6164.

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Flow Cytometry Analysis of Socs1flox Mice

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Blood (50μl) was collected weekly 24 h after OXA application. Whole blood samples were processed using lysis buffer (from Hospital Pharmacy at LUMC) for 10 min at 37 °C. Cells were incubated with monoclonal antibodies for 30 min on ice.
Fluorescence-labeled antibodies included anti-mouse CD3 (clone 145-2C11, BD, The Netherlands), anti-mouse CD19 (clone 1D3, Thermo Fisher Scientific, The Netherlands), anti-mouse CD4 (clone RM4-5, Thermo Fisher Scientific, The Netherlands), anti-mouse CD8 (clone 53–6.7, Biolegend, The Netherlands) and anti-ΔhCD4 (clone RPA-T4, eBioscience™, The Netherlands). Of note, the antibody for ΔhCD4 should be specific for this fragment of human-CD4 as a reporter in the Socs1flox transgenic mouse. Samples were processed in a BD Fortessa flow cytometer and analyzed using the FlowJo software.

Luo Y., Vermeer M.H., de Haan S., Kinderman P., de Gruijl F.R., van Hall T, & Tensen C.P. (2023). Socs1-knockout in skin-resident CD4+ T cells in a protracted contact-allergic reaction results in an autonomous skin inflammation with features of early-stage mycosis fungoides. Biochemistry and Biophysics Reports, 35, 101535.

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Generation of HLA-DR4 and HLA-A2 Mice

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The DRAG mice (NOD.HLA-DR4.RagKO.IL2RgcKO) express HLA-DR4 molecules in NRG background, and they have been previously described12 (link). The DRAG mice were crossed with HLA-A2.1.NOD mice (stock #006611, The Jackson Laboratory) to generate F1 mice that were intercrossed to generate F2 mice. The F2 progeny was screened for expression of HLA-DR4 and HLA-A2 molecules by FACS using peripheral white blood cells stained with HLA-DR and A2 antibodies (clones #Tu39 and #BB7.2, BD Biosciences, San Jose, CA). Rag1KO litters were identified by the absence of mouse T cells upon staining of peripheral white blood cells with anti-mouse CD3 (clone #145-2C11, BD Biosciences). The IL2RγcKO mutation was screened by PCR as previously described12 (link). Mice were bred at the Veterinary Program Service at NMRC/WRAIR.

Majji S., Wijayalath W., Shashikumar S., Pow-Sang L., Villasante E., Brumeanu T.D, & Casares S. (2016). Differential effect of HLA class-I versus class-II transgenes on human T and B cell reconstitution and function in NRG mice. Scientific Reports, 6, 28093.

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Quantitative Analysis of Vav1 Expression

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RNA was extracted from thymocytes and splenocytes of 4 week old vav-CAR mice and WT mice using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. Inguinal lymph nodes from 6–8 week old vav-CAR mice were harvested and dissociated in PBS through a 70 μm filter to form a single cell suspension. Cells were cultured overnight in RPMI plus additives in the presence or absence of anti-mouse CD3 (clone 145-2C11 at 500 ng/ml) and anti-mouse CD28 (clone 37.51 at 500 ng/ml) (BD Bioscience). The cells were harvested the next day and RNA extracted as above. Extracted RNA was converted to cDNA using random primers. Quantitative Real Time PCR was performed on cDNA using primers specific for the Vav1 mRNA transcript (Primer code: Hs01041613_m1, Life Technologies). Mouse β-actin primers were used as controls (Primer 4352933E, Life Technologies). The QRTPCR was run according to the manufacturer’s instructions using the StepOne System (Life Technologies)

Yong C.S., Westwood J.A., Schröder J., Papenfuss A.T., von Scheidt B., Moeller M., Devaud C., Darcy P.K, & Kershaw M.H. (2015). Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice. PLoS ONE, 10(10), e0140543.

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Quantifying IFN-γ Responses and T-cell Subsets

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The IFN-γ-secreting splenocytes were measured at 7, 17, and 45 dpi using a mouse IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (BD Life Sciences, USA). Briefly, 5 × 10⁵ splenocytes/well were stimulated with Z_EDIII (1 μg/ml). Spots were counted under a dissecting microscope (Olympus, model no. SZH-ILLB). To measure secreted cytokines, splenocytes (5 × 10⁵/well) were stimulated with Z_EDIII (1 μg/ml) for 48 h and the supernatant of splenocytes was collected. The levels of cytokines were determined using mouse tumor necrosis factor (TNF)-α ELISA MAX™ standard set, mouse IFN-γ ELISA MAX™ standard set, and mouse interleukin (IL)-12 ELISA MAX™ standard set (BioLegend, USA). Each assay was performed in triplicate. To analyze T lymphocyte subtypes, splenocytes were stained with the following antibodies at a dilution of 1:200 with 1% BSA in PBS at 4 °C for 1 h: anti-mouse CD3 (clone 145-2C11, BD Biosciences), and anti-mouse CD4 (clone RM4-5, BD Biosciences) or anti-mouse CD8 (clone 53–6.7, BD Biosciences) as described previously [27 (link)]. Cell phenotype was analyzed using a FACS Calibur flow cytometer (Becton Dickinson).

Shin M., Kim K., Lee H.J., Jung Y.J., Park J, & Hahn T.W. (2022). Vaccination with a Zika virus envelope domain III protein induces neutralizing antibodies and partial protection against Asian genotype in immunocompetent mice. Tropical Medicine and Health, 50, 91.

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BTLA Regulation in T Cell Activation

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Murine OT-1 BTLA KO cells overexpressing BTLA WT or mutants constructs were re-stimulated with either 10 ng/ml anti-mouse CD3 (Clone 145-2C11, BD Pharmingen™) alone or with recombinant mouse HVEM Fc (R&D systems) plate-bound for 8 h prior to harvest with the cell lysis buffer (kindly provided by RPPA core facility at The University of Texas M.D. Anderson Cancer Center). The cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4°C. The protein supernatant was quantified using protein assay kit (Thermo scientific). RPPA was processed and normalized as previously described (20 (link)). Differential fold expression of protein was analyzed using Linear models and empirical Bayes methods(21 (link)). Volcano plots were generated using R system. For human TIL, four TIL lines were stained with anti-CD8 (clone RPA-T8, BD Pharmingen™), anti-BTLA (clone J168, BD Pharmingen™), and Sytox blue (Molecular Probe™) under aseptic condition. The cells were sorted based on expression of CD8⁺BTLA⁺ using FACSAria (BD Biosciences). On the next day, sorted TIL were re-stimulated with anti-human CD3 (clone OKT-3, eBioscience) with or without recombinant human HVEM-Fc (R&D systems) plate-bound for 8 h prior to harvest with the cell lysis buffer. The protein samples were processed, normalized, and analyzed as similar to the mouse experiment described above.

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