Human t activator cd3 cd28

Manufactured by Thermo Fisher Scientific

Sourced in Norway

The Human T-Activator CD3/CD28 is a laboratory product designed to activate and expand human T cells. It consists of beads coated with antibodies that bind to the CD3 and CD28 receptors on the surface of T cells, providing the necessary signals for T cell activation and proliferation.

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Lab products found in correlation

Interleukin 2 (il 2), by Miltenyi Biotec (5 mentions) Pan t cell isolation kit, by Miltenyi Biotec (4 mentions) X vivo 15 medium, by Lonza (4 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (3 mentions) Human serum, by Gemini Bio (3 mentions) Dynabead, by Thermo Fisher Scientific (3 mentions) X vivo 15 media, by Lonza (2 mentions) Cts immune cell sr, by Thermo Fisher Scientific (2 mentions) Macs ld column, by Miltenyi Biotec (2 mentions) X vivo 15, by Lonza (2 mentions) Anti il 4, by R&D Systems (1 mentions) Non essential amino acids (neaa), by Merck Group (1 mentions) Hepes, by Solarbio (1 mentions) Transforming growth factor beta1 tgf β1, by R&D Systems (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) β mercaptoethanol, by Merck Group (1 mentions) Tgf β1, by R&D Systems (1 mentions) Penicillin, by Solarbio (1 mentions) Streptomycin, by Solarbio (1 mentions)

Close spelling variants of this product

Dynabead human T-activator CD3/CD28 ImmunoCult Human CD3/CD28/CD2 T Cell Activator Anti-CD3/CD28 Human T-Activator Dynabeads Dynabeads Human T-Activator CD3/CD28 beads Dynabeads Human T-Activator CD3/CD28/CD137 Gibco Dynabeads Human T-Activator CD3/CD28 Human T-Activator CD3/CD28 beads Dynabeads™ Human T-Activator CD3/CD28 kit Anti-CD3/anti-CD28 human T-Activator Dynabeads Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation Kit

12 protocols using human t activator cd3 cd28

Th17 and Treg Cell Polarization from Activated CD4+ T Cells

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The isolated naïve CD4⁺ T cells were cultured in 96-well, U-bottomed plates at a concentration of 1 × 10⁵ cells/well. Human T-Activator CD3/CD28 (Gibco, Norway) was added in culture medium overnight to activate CD4⁺ T cells. For Th17 cell polarization, activated CD4⁺ T cells were cultured with 20 ng/mL interleukin-6 (IL-6) (R&D Systems), 2.5 ng/mL transforming growth factor beta-1 (TGF-β1) (R&D Systems), 2 μg/mL anti-IL-4 (R&D Systems), and 2 μg/mL anti-interferon-gamma (IFN-γ) (Invitrogen). For Treg cell polarization, activated CD4⁺ T cells were cultured with TGF-β1 (2.5 ng/mL, R&D systems) and IL-2 (10 ng/mL; BD Biosciences). Culture medium consisted of RPMI 1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Australia), 2 mM L-glutamine (Solarbio, China), 1 mM sodium pyruvate (Solarbio), 50 μM beta-mercaptoethanol (Sigma-Aldrich, USA), 1× nonessential amino acids (Sigma-Aldrich), 100 U/mL penicillin (Solarbio), 100 mg/mL streptomycin (Solarbio) and 10 mM Hepes (Solarbio) [20 (link), 21 (link)].

Jiao A., Yang Z., Fu X, & Hua X. (2020). Phloretin Modulates Human Th17/Treg Cell Differentiation In Vitro via AMPK Signaling. BioMed Research International, 2020, 6267924.

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Flow Cytometry and Immunoblotting for T-cell Analysis

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Flow cytometric antibodies - anti-CD3, CD4, CD8, CD19 and CD138 antibodies (cat #100220, 100509, 100711, 115505, 142505 BioLegend). Immunohistochemistry antibodies - anti-CD3 antibody [CD3-12] (ab11089, Abcam) and anti-F4/80 (cat # 70076, Cell Signaling). DSA analysis antibodies - F(ab)2’-IgM-APC (Jackson Immunoresearch Laboratories) and F(ab)2’-IgG-FITC (cat # F11021, Thermo Fisher Scientific). Western blot antibodies - GAPDH antibody (ThermoFisher cat# MA5-15738); IκB and p32/36 IκB (ThermoFisher cat# MA5-15087, Cell Signaling cat# 4814T); p100/p52, p105/p50 and p65/RelA (Cell Signaling cat #55764). MILLIPLEX MAP Mouse High Sensitivity T Cell Magnetic Bead Panel (Millipore, MHSTCMAG-70K). NU7441 (Selleckchem, S2638). PMA (AAJ63916MCR), PHA (NC1293968) and ionomycin (BP25271), Fisher Scientific. Human T-Activator CD3/CD28 (Gibco, Cat # 11161D). Jurkat T cells (ATCC, Clone E6-1, cat # TIB-152)

Harrison D.K., Waldrip Z.J., Burdine L., Shalin S.C, & Burdine M.S. (2021). DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival. Transplantation, 105(3), 540-549.

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Cryopreserved PBMC CD22-CAR T-cell Generation

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Cryopreserved human PBMCs (ALLCELLS) were thawed and plated at a density of 1 × 10⁶ cells/ml in X-vivo-15 media (Lonza) supplemented with 5% human AB serum (Gemini) or CTS Immune Cell SR (Thermo Fisher Scientific) and 20 ng/ml IL-2 (Miltenyi Biotech) for an overnight culture at 37°C. The next day, the PBMCs were activated using human T activator CD3/CD28 (Life Technology) in serum-free X-vivo-15 media without IL-2. One million activated PBMCs (in 600 μl) were immediately incubated without removing the beads in an untreated 12-well plate pre-coated with 30 μg/ml Retronectin (Takara) in the presence of lentiviral particles encoding the CD22 targeting CAR for 2 h at 37°C. Six hundred microliters of 2× X-vivo-15 media (X-vivo-15, 10% human AB serum and 40 ng/ml IL-2) was added after 2 to 3 h, and the cells were incubated at 37°C for 72 h.

Juillerat A., Tkach D., Yang M., Boyne A., Valton J., Poirot L, & Duchateau P. (2020). Straightforward Generation of Ultrapure Off-the-Shelf Allogeneic CAR-T Cells. Frontiers in Bioengineering and Biotechnology, 8, 678.

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SWIFF-CAR T Cell Engineering Protocol

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Human PBMCs () were thawed and plated at 1 × 10⁶ cells/ml in X-vivo-15 media (Lonza) supplemented with 5% hAB serum (Gemini) or CTS Immune Cell SR (ThermoFisher) and 20 ng/ml IL-2 (Miltenyi Biotech) for overnight culture at 37 °C. The next day, the PBMCs were activated using human T activator CD3/CD28 (Life Technology) in serum-free X-vivo-15 media without IL-2. One million activated PBMCs (in 600 μl) were immediately incubated without removing the beads in an untreated 12-well plate pre-coated with 30 μg/ml retronectine (Takara) in the presence of lentiviral particles encoding the engineered SWIFF-CAR for 2 h at 37 °C. Six hundred microliters of 2x X-vivo-15 media (X-vivo-15, 10% hAB serum and 40 ng/ml IL-2) was added after 2 to 3 h, and the cells were incubated at 37 °C for 72 h. If required, transduced T-cells were then expanded for 11 days in G-Rex10 (Wilson Wolf) in 40 ml of complete X-vivo-15 media.

Juillerat A., Tkach D., Busser B.W., Temburni S., Valton J., Duclert A., Poirot L., Depil S, & Duchateau P. (2019). Modulation of chimeric antigen receptor surface expression by a small molecule switch. BMC Biotechnology, 19, 44.

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Detailed Protocol for CAR-T Cell Therapy

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Cell culture reagents, X-vivo-15 was obtained for Lonza (cat#BE04-418Q), IL-2 from Miltenyi Biotech (cat#130-097-748), human serum AB from Seralab (cat#GEM-100–318), human T activator CD3/CD28 from Life Technology (cat#11132D), CD34 MicroBead kit and MACS® LD-column from Miltenyi Biotech (cat#130-046-702 and cat#130-042-901 respectively), QBEND10-APC from R&D Systems (cat#FAB7227A), Rituximab from internal sources, Baby rabbit complement (cat#C12CA) from AbD Serotec/BIO-RAD, eFluor 780 from eBioscience (cat# 65-0865-14) and retronectine form Takara (cat#T100B). Cryopreserve human PBMC, acquired from Allcells (cat#PB006F) were used in accordance with all applicable Pfizer policies, including IRB/IEC approval.

Valton J., Guyot V., Boldajipour B., Sommer C., Pertel T., Juillerat A., Duclert A., Sasu B.J., Duchateau P, & Poirot L. (2018). A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies. Scientific Reports, 8, 8972.

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Engineered Cell Lines for Research

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HEK293T (ATCC) and MV4-11 (ATCC) were cultured in media per the supplier’s recommendation. Expi293 (Thermo Fisher Scientific) was cultured in media per the supplier’s recommendation. U87-EGFR is a kind gift from Cellectis SA (Paris, France). U87-EGFR was derived from the parental cell line, U87MG (ATCC) by first knocking out endogenous EGFR using Transcription Activator-Like Effector Nucleases (TALEN), and then stably overexpressing full-length human EGFR via lentiviral transduction. X-VIVO^TM 15 was obtained from Lonza, rhIL-2 from Miltenyi Biotech, human serum AB from Seralab, human T-activator CD3/CD28 from Thermo Fisher Scientific, MACS^® LD-column from Miltenyi Biotech, Hyclone FBS from GE Healthcare Life Sciences, Ficoll-Paque PLUS from GE Healthcare Life Sciences and Pan T cell Isolation Kit, human from Miltenyi Biotech. Methotrexate disodium salt and leucovorin calcium were obtained from Alfa Aesar and Sigma, respectively.

Park S., Pascua E., Lindquist K.C., Kimberlin C., Deng X., Mak Y.S., Melton Z., Johnson T.O., Lin R., Boldajipour B., Abraham R.T., Pons J., Sasu B.J., Van Blarcom T.J, & Chaparro-Riggers J. (2021). Direct control of CAR T cells through small molecule-regulated antibodies. Nature Communications, 12, 710.

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Isolation and Activation of Primary Human T Cells

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Buffy coats from healthy volunteer donors were obtained from the New York Blood Center. Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and typically frozen in 90% FBS /10% DMSO. Frozen PBMCs were cultured for 1h in T cell medium (see below; >85% viability) and then used for T cell isolation. T lymphocytes were purified using the Pan T cell isolation kit (Miltenyi Biotech). T cells (>90% viability) were activated with Dynabeads (1:1 beads:cell) Human T-Activator CD3/CD28 (ThermoFisher) in X-vivo 15 medium (Lonza) supplemented with 5% human serum (Gemini Bioproducts) with 5 ng/ml human recombinant IL7 (Peprotech) and 5 ng/ml human recombinant IL15 (Peprotech) at a density of 10⁶ cells per ml. The medium was changed every 2 days, and cells were replated at 1-1.5 x10⁶ cells per ml.

Mansilla-Soto J., Eyquem J., Haubner S., Hamieh M., Feucht J., Paillon N., Zucchetti A.E., Li Z., Sjöstrand M., Lindenbergh P.L., Saetersmoen M., Dobrin A., Maurin M., Iyer A., Angus A.G., Miele M.M., Zhao Z., Giavridis T., van der Stegen S.J., Tamzalit F., Rivière I., Huse M., Hendrickson R.C., Hivroz C, & Sadelain M. (2022). HLA-independent T cell receptors for targeting tumors with low antigen density. Nature medicine, 28(2), 345-352.

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Isolation and Activation of T Cell Subsets

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Blood of donor was obtained from the Blood Donor Center at Boston Children’s Hospital (Boston, MA) as approved by the University Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) were isolated from blood using Lymphoprep (STEMCELL Technologies, 07851) and activated with Human T-activator CD3/CD28 (Thermo Fisher, 11131D) for further lentiviral transduction. CD4+ T cell and CD8+ T cell were isolated from blood using RosetteSep Human T cell enrichment cocktail (STEMCELL Technologies, 15022 and 15023), and T reg cells were isolated using the EasySep human CD4+CD127lowCD25+ regulatory T cell isolation kit (STEMCELL Technologies, 18063) according to the manufacturer’s protocol.

Li H.S., Wong N.M., Tague E., Ngo J.T., Khalil A.S, & Wong W.W. (2022). High-performance multiplex drug gated CAR circuits. Cancer cell, 40(11), 1294-1305.e4.

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Expansion of Human T Lymphocytes

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De-identified buffy coats from healthy volunteer donors were purchased from the New York Blood Center (IRB exempted). Peripheral blood mononuclear cells were isolated by density gradient centrifugation, and T lymphocytes were then purified using the Pan T cell isolation kit (Miltenyi Biotech). Cells were activated with Dynabeads (1:1 beads:cell) Human T-Activator CD3/CD28 (ThermoFisher) in X-vivo 15 medium (Lonza) supplemented with 5% human serum (Gemini Bioproducts) with 100 U/ml IL-2 (Miltenyi Biotech) at a density of 10⁶ cells/ml. The beads were removed by magnetic separation 48 hr after activation. The medium was changed every 2 days, and cells were replated at 10⁶ cells/ml.

Perna F., Berman S., Soni R., Mansilla-Soto J., Eyquem J., Hamieh M., Hendrickson R., Brennan C, & Sadelain M. (2017). Integrating proteomics and transcriptomics for systematic combinatorial chimeric antigen receptor therapy of AML. Cancer cell, 32(4), 506-519.e5.

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Modulation of Primary T Cell Function

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Recombinant human interleukin (IL)-2 was purchased from R&D Systems. For manipulation of human primary T cells, the Dynabeads Untouched Human T cell kit and Human T-Activator CD3/CD28 were purchased from Thermo Fisher Scientific, Inc. For western blot analysis, anti-RFP/mCherry rabbit polyclonal antibody (PM005; 1:1,000 dilution) was purchased from Medical and Biological Laboratories; anti-GFP/YFP rabbit polyclonal antibody (SC-8334; 1:5,000 dilution) and anti-syntaxin 4 mouse mAb (QQ-17; 1:1,000 dilution) were purchased from Santa Cruz Biotechnology, Inc.; anti-β-actin mouse mAb (A1978; 1:10,000 dilution) was purchased from Sigma Aldrich; Merck KGaA; anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (NA934; 1:5,000 dilution) and anti-mouse HRP conjugated antibody (NA9310; 1:5,000 dilution) were purchased from GE Healthcare. For flow cytometric analysis, PE anti-human CD69 antibody (FN50; 1:20 dilution), PerCP/Cy5.5 anti-human HER2 antibody (24D2; 1/50 dilution), and FITC anti-human CD19 antibody (4G7; 1/50 dilution) were purchased from BioLegend, Inc. Hoechst 33258 was purchased from Dojindo Molecular Technologies, Inc. HIV protease (HIVPR) inhibitors, saquinavir and nelfinavir, were kindly provided by Professor Yamaoka at the Department of Virology, TMDU (Tokyo, Japan).

Aoyama S., Yasuda S., Li H., Watanabe D., Umezawa Y., Okada K., Nogami A., Miura O, & Kawamata N. (2022). A novel chimeric antigen receptor (CAR) system using an exogenous protease, in which activation of T cells is controlled by expression patterns of cell-surface proteins on target cells. International Journal of Molecular Medicine, 49(4), 42.

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