Anti human cd3 clone okt3

Manufactured by Thermo Fisher Scientific

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The Anti-human CD3 (clone OKT3) is a monoclonal antibody that binds to the CD3 complex on the surface of human T cells. It recognizes the epsilon chain of the CD3 complex.

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Lab products found in correlation

Anti human cd28 clone cd28, by Thermo Fisher Scientific (5 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions) Tj 6 centrifuge, by Beckman Coulter (2 mentions) Vi cell xr, by Beckman Coulter (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions) Anti cd4 clone a161a1, by BioLegend (1 mentions) Anti cd28 37, by BioLegend (1 mentions) Anti cd8 rm2206, by BioLegend (1 mentions) Anti cd3ε 145 2c11, by BD (1 mentions) Anti pdpn clone nz 1, by Thermo Fisher Scientific (1 mentions) Anti thy1 clone 5e10, by BD (1 mentions) Vi cell xr cell viability analyzer, by Beckman Coulter (1 mentions) Human serum, by Merck Group (1 mentions) Amphotericin b, by Thermo Fisher Scientific (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Elisa, by BD (1 mentions) Methyl 3h thymidine, by PerkinElmer (1 mentions) Recombinant tgf β1, by R&D Systems (1 mentions) Cell lysis buffer, by Thermo Fisher Scientific (1 mentions) Phosphorylated erk1 2, by Cell Signaling Technology (1 mentions)

9 protocols using anti human cd3 clone okt3

Comprehensive Murine Tissue Analysis Panel

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For flow cytometry and imaging studies with murine tissues, the following antibodies were purchased from Biolegend: anti-CD45 (30-F11), anti-CD31 (390), anti-PDPN (8.1.1), anti-α-smooth muscle actin (1A4), anti-Thy1 (53–2.1), anti-CD140α (APA5), anti-CD140b (APB5), anti-CD106 (429), anti-CD4 (RM4.5), and anti-CD8 (RM2206). The anti-FAP used for flow cytometry analysis was provided by Ellen Pure and has been validated (anti-mouse FAP 73.3)(8 (link)). For immunofluorescent analysis, the Roche anti-FAP 28H1 clone was used (26 (link)). Functional-grade purified anti-CD3ε (145–2C11) was from BD Biosciences, and anti-CD28 (37.51) was from Biolegend. Antibodies used to stain human samples include viability dye (L10119; Life Technologies), anti-PDPN (clone NZ1.3; eBioscience), anti-Thy1 (clone 5E10; BD Biosciences), anti-CD4 (clone A161A1; Biolegend) and anti-CD8 (clone HIT8α; Biolegend). For PBMC stimulation, anti-human CD3 (clone OKT3; eBioscience) and anti-human CD28 (clone CD28.2; eBioscience) were used. L-NMMA (NG-monomethyl-L-arginine) was purchased from Calbiochem and CFSE (carboxyfluorescein diacetate succinimidyl ester) was from Invitrogen.

Cremasco V., Astarita J., Grauel A.L., Keerthivasan S., MacIsaac K., Woodruff M.C., Wu M., Spel L., Santoro S., Amoozgar Z., Laszewski T., Migoni S.C., Knoblich K., Fletcher A.L., LaFleur M., Wucherpfennig K.W., Pure E., Dranoff G., Carroll M.C, & Turley S.J. (2018). FAP delineates heterogeneous and functionally divergent stromal cells in immune-excluded breast tumors. Cancer immunology research, 6(12), 1472-1485.

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T Cell Activation and Cytokine Analysis

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Fresh whole blood samples collected in heparin were diluted tenfold with RPMI and 90 µl of the diluted blood was incubated in 96-well flat-bottom tissue culture plates in a humidified incubator at 5% CO₂, 37ºC for 2 days in the presence or absence of anti-CD3 + anti-CD28. Specifically, to stimulate the T cells the plates were pre-coated overnight with 2 µg/mL of anti-human CD3 (clone OKT3, eBioscience, San Diego, CA). Anti-human CD28 (clone CD28.2, eBioscience, San Diego, CA) was then added at a final concentration of 1 µg/mL to each well. Alternatively, PBMCs isolated as for immunophenotyping above were stimulated anti-CD3 and anti-CD28 as previously described^{21 (link)}. PBMCs were counted using a Vi-Cell XR cell viability analyzer (Beckman Coulter, Brea, CA) and resuspended at 10⁶ cells/mL in RPMI + 10% autologous plasma + 100 U/mL penicillin/streptomycin. The PBMCs were aliquoted (50 μl/well) into 96-well flat-bottom tissue culture plates pre-coated or not as above with 0.5 µg/mL of anti-human CD3 and then 2 µg/mL anti-human CD28 added. After four days of incubation at 37 ˚C, 5% CO₂, the plates were then centrifuged at 300 × g in a Beckman TJ-6 centrifuge for 5 min and the supernatants collected for IFNγ analysis.

Elisia I., Lam V., Cho B., Hay M., Li M.Y., Yeung M., Bu L., Jia W., Norton N., Lam S, & Krystal G. (2020). The effect of smoking on chronic inflammation, immune function and blood cell composition. Scientific Reports, 10, 19480.

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Isolation and Expansion of Tumor-Infiltrating Lymphocytes

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Lesion tissue was dissected into small pieces (2 × 2 mm) and transferred into 24-well tissue culture-treated plates at three pieces per well in 2 ml human tumour-invading lymphocyte (TIL) medium (RPMI1640 (Pan Biotec) with 10% human serum (Sigma Aldrich), 2 mM l-glutamine, 1.25 μg/ml amphotericin B (both Gibco), 1,000 U/ml IL-2 (Proleukin)) containing 30 ng/ml anti-human CD3 (clone OKT-3, eBioscience). Medium was exchanged every 2–3 days and tissue pieces removed on day 7. LILs that migrated out of the tumour into the medium were further expanded until day 14 and cryopreserved as above.

Platten M., Bunse L., Wick A., Bunse T., Le Cornet L., Harting I., Sahm F., Sanghvi K., Tan C.L., Poschke I., Green E., Justesen S., Behrens G.A., Breckwoldt M.O., Freitag A., Rother L.M., Schmitt A., Schnell O., Hense J., Misch M., Krex D., Stevanovic S., Tabatabai G., Steinbach J.P., Bendszus M., von Deimling A., Schmitt M, & Wick W. (2021). A vaccine targeting mutant IDH1 in newly diagnosed glioma. Nature, 592(7854), 463-468.

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Activation and IFNγ Analysis of PBMCs

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PBMCs isolated as above were counted using a Vi-Cell XR cell viability analyzer (Beckman Coulter, Brea, CA) and resuspended at 10⁶ cells/mL in RPMI + 10% autologous plasma + 100 U/mL penicillin/streptomycin. The PBMCs were aliquoted (50 μl/well) into 96-well flat-bottom tissue culture plates that were pre-coated overnight with 0.5 μg/mL of anti-human CD3 (clone OKT3, eBioscience, San Diego, CA). Anti-human CD28 (clone CD28.2, eBioscience, San Diego, CA) at a final concentration of 2 μg/mL was then added to each well, and the plates incubated in a humidified incubator at 5% CO₂, 37ºC for 4 days. The plates were then centrifuged at 300 x g in a Beckman TJ-6 centrifuge for 5 min and the supernatants collected for IFNγ analysis by ELISA (BD Biosciences, San Diego, CA).

Elisia I., Lam V., Cho B., Hay M., Li M.Y., Kapeluto J., Elliott T., Harris D., Bu L., Jia W., Leung H., Mohn W, & Krystal G. (2020). Exploratory examination of inflammation state, immune response and blood cell composition in a human obese cohort to identify potential markers predicting cancer risk. PLoS ONE, 15(2), e0228633.

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PBMC stimulation and cytokine/proliferation analysis

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PBMCs isolated as above were counted using a Vi-Cell XR cell viability analyzer (Beckman Coulter, Brea, CA) and resuspended at 10⁶ cells/mL in RPMI + 10% autologous plasma + 100 U/mL penicillin/streptomycin. The PBMCs were aliquoted (50 μl/well) into 96-well flat-bottom tissue culture plates that were pre-coated overnight with 0.5 μg/mL of anti-human CD3 (clone OKT3, eBioscience, San Diego, CA). Anti-human CD28 (clone CD28.2, eBioscience, San Diego, CA) at a final concentration of 2 μg/mL was then added to each well, and the plates incubated in a humidified incubator at 5% CO₂, 37°C for 4 days. The plates were then centrifuged at 300 x g in a Beckman TJ-6 centrifuge for 5 min and the supernatants collected for IFNγ analysis. Identical plates were pulse-labeled with [methyl-³H]-thymidine (1 μCi/well, 2 Ci/mmol, PerkinElmer, Woodbridge, ON) for 24 hrs. Cells were harvested and the incorporated ³H-thymidine quantified using a LKB Betaplate Harvester and Liquid Scintillation Counter (LKB Wallac).

Elisia I., Lam V., Hofs E., Li M.Y., Hay M., Cho B., Brooks-Wilson A., Rosin M., Bu L., Jia W, & Krystal G. (2017). Effect of age on chronic inflammation and responsiveness to bacterial and viral challenges. PLoS ONE, 12(11), e0188881.

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T Cell Proliferation and Cytokine Assay

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CD4⁺ and CD8⁺ T cells were labeled with CFSE as described earlier and divided into aliquots of 2 × 10⁵ cells/well into a 96-well tissue culture plate pre-coated with anti-human CD3, clone OKT3 (2.5 μg/ml, eBioscience) or with PBS as a control. Recombinant TGF-β-1 (R&D Systems) diluted in PBS was added at the concentrations indicated in the figure legends and cells were cultured at 37°C for the times indicated in the legends. Cell culture medium was collected for the measurement of secreted IFN-γ with the human IFN-γ ELISA Ready-SET-Go! kit (eBioscience). Flow cytometry was used to measure the extent of CFSE dilution and for intracellular staining of granzyme B as described earlier.

Newman D.K., Fu G., Adams T., Cui W., Arumugam V., Bluemn T, & Riese M.J. (2016). The adhesion molecule PECAM-1 enhances the TGFβ-mediated inhibition of T cell function. Science signaling, 9(418), ra27.

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T Cell Activation Signaling Pathway

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Mouse immunoglobulin G (IgG) anti-human pure CD3 (clone UCHT1), ionomycin, goat anti-mouse-IgG (GAM), sodium fluoride (NaF), sodium orthovanadate, anti-protease cocktail and Bradford reagent were purchased from Sigma, USA. Fura-2-acetoxymethyl ester (Fura-2/AM) was procured from Calbiochem, USA. Cell lysis buffer was purchased from Invitrogen, USA. Anti-human CD3 (clone OKT-3) and anti-human CD28 (clone CD28.2) were procured from eBioscience, USA. Phosphorylated ZAP-70, phosphorylated ERK1/2, phosphorylated p38 MAPK, phosphorylated protein kinase C theta (PKC-θ), β-Actin and goat polyclonal IgG anti-mouse peroxidase-conjugated antibodies were procured from Cell Signaling Technology (CST), USA. Enhanced chemiluminescence (ECL) reagents were procured from Millipore, USA. Lyophilized M. tuberculosis antigens (ESAT-6 and Ag85A) were procured from the BEI Research Resources Repository funded by the National Institute of Allergy and Infectious Diseases and managed by ATCC, USA. PPD RT-49 (Research Tuberculin) was procured from Statens Serum Institut, Denmark. All antigens were dissolved in filtered phosphate-buffered saline (PBS) at 7.4 pH to make a 1 mg/ml concentration.

Sharma B., Rathour D., Uddin S., Joshi B., Chauhan D.S, & Kumar S. (2022). Exploring modulations in T-cell receptor-mediated T-cell signaling events in systemic circulation and at local disease site of patients with tubercular pleural effusion: An attempt to understand tuberculosis pathogenesis at the local disease site. Frontiers in Medicine, 9, 983605.

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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.

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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Quantifying T-Cell Actin Cytoskeleton

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8-well chamber culture slides (BD Falcon, San Jose, CA, USA) were coated with 0.01% poly-L-lysine solution (Sigma-Aldrich) for 15 minutes at room temperature and subsequently with 10µg/ml anti-human CD3 (clone OKT3; eBioscience) overnight at 4°C. 0.5×10 6 (for 1 minute of spreading) or 0.2×10 6 (for 3 and 5 minute of spreading) cells were plated gently onto the slides and allowed to spread for 1, 3 or 5 minutes. Cells were fixed with 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes and blocked with 5% goat serum in 10% FBS/0.02% sodium azide in PBS for an hour. Cells were then stained with rhodamine phalloidin (Thermo Fisher Scientific)
according to the manufacturer's instructions. After washing, the cell specimens were mounted with Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Slides were analyzed using Leica DM4500B fluorescence microscope (Leica Microsystems, Wetzlar, Germany) with a 100× oil objective. Images were acquired using a Leica DFC365 FX monochrome digital camera and Leica LAS AF software. Percentage of spreading was calculated by counting at least 100 cells per experimental condition. Cells that form an F-actin ring at the cell periphery were scored as positive while cells without an Factin ring were scored as negative.

Lim S.P., Ioannou N., Ramsay A.G., Darling D., Gäken J, & Mufti G.J. (2018). miR-181c-BRK1 axis plays a key role in actin cytoskeleton-dependent T cell function. Journal of leukocyte biology, 103(5).

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