Anti cd3 cd28 mab

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

Anti-CD3/CD28 mAbs are monoclonal antibodies that recognize and bind to the CD3 and CD28 proteins on the surface of T cells. These antibodies are commonly used in cell culture and immunology research to activate and expand T cells in vitro.

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Lab products found in correlation

Tgf β1, by Thermo Fisher Scientific (2 mentions) Retronectin, by Takara Bio (1 mentions) Recombinant human interleukin 2 il 2, by Thermo Fisher Scientific (1 mentions) Intracellular fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions) Facsaria 2, by BD (1 mentions) Anti cd3 microbeads, by Miltenyi Biotec (1 mentions) Interleukin 2 (il 2), by R&D Systems (1 mentions) Magnisort human t cell enrichment kit, by Thermo Fisher Scientific (1 mentions) Anti il 17a pe mab, by BD (1 mentions) Il 1β, by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Anti mouse foxp3 pe or isotype control mabs, by BD (1 mentions) Cd4 cd25 regulatory t cell isolation kit, by Miltenyi Biotec (1 mentions)

Close spelling variants of this product

Anti-CD3 (512 citations) Anti-CD28 (306 citations) Anti-CD3/CD28 (45 citations) Anti-CD28 mAb (18 citations) Anti-CD3 mAb (12 citations) Anti-CD3 and anti-CD28 mAbs (3 citations) Anti-CD3/anti-CD28 mAb-coated Human T-Expander beads (3 citations) Anti-CD3/CD28 mAb-coated Dynabeads (3 citations) Anti-CD3 and anti-CD28 mAbs-coated beads (2 citations) Anti-CD3/anti-CD28 mAb-coated microbeads (2 citations)

6 protocols using anti cd3 cd28 mab

Generation of CLL-1 CAR-T Cells

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The DNA sequence of the scFv derived from the CLL-1 antibody was synthesized and cloned into the lenti-vector pELNS. CD28, 4-1BB, and CD3-ζ signaling domains were then constructed into to generate the CLL-1 CAR. Thy1.1 was constructed into as a reporting marker. The lentiviral supernatants were produced by transfecting 293 T cells and concentrated by ultra-centrifuging. The concentrated CLL-1 CAR lenti-virus was immediately stored at − 80 °C for further use.
Healthy donor-derived peripheral blood mononuclear cells (PBMCs) or CD3⁺ enriched T cells (Miltenyi) were expanded in vitro using anti-CD3/CD28 mAbs (Ebioscience) and recombinant human interleukin-2 (IL-2) at 20 ng/ml (Peprotech) for 48 h. The activated T cells were then transduced with lentiviral supernatants on day 3 in plates pre-coated with retronectin (Takara). After transduction, T cells were cultured with IL-2, IL-7, and IL-15. CAR expression on T cells was measured 72 h later, and the cells were isolated by immunomagnetic selection using a biotinylated-Thy1.1 antibody followed by a secondary stain with anti-biotin magnetic beads (Biolegend).

Wang J., Chen S., Xiao W., Li W., Wang L., Yang S., Wang W., Xu L., Liao S., Liu W., Wang Y., Liu N., Zhang J., Xia X., Kang T., Chen G., Cai X., Yang H., Zhang X., Lu Y, & Zhou P. (2018). CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia. Journal of Hematology & Oncology, 11, 7.

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Β1-Adrenoceptor Regulation of Treg Cells

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CD4⁺ T cells sorted from the splenocytes of healthy mice were with or without preactivation by anti-CD3/CD28 mAbs (1 μg/mL) (eBioscience, USA) for 72 h. Then, the cells were stimulated with different concentrations of β₁-AA (10⁻⁸, 10⁻⁷, or 10⁻⁶ mol/L), with or without metoprolol (the β₁-AR-specific blocker, 10⁻⁸ mol/L), for 24 h. Subsequently, CD4⁺ T cells were surface stained with anti-CD4/CD25 mAbs (FITC/phycoerythrin (PE)) for 30 min at 4 °C. After staining, cells were fixed and permeabilized using an intracellular fixation and permeabilization buffer set (eBioscience, USA), followed by intracellular staining with anti-FoxP3 mAb (BV421) (BD Bioscience, USA). The stained cells were centrifuged at 1500 rpm for 5 min, and re-suspended in 250 μL flow cytometric buffer solution (1% FBS in phosphate-buffered saline (PBS)). Flow cytometry was performed on the FACS Aria II flow cytometer (Becton, Dickinson and Company).

Xu W., Wu Y., Wang L., Bai Y., Du Y., Li Y., Cao N., Zhao Y., Zhang Y, & Liu H. (2019). Autoantibody against β1-adrenoceptor promotes the differentiation of natural regulatory T cells from activated CD4+ T cells by up-regulating AMPK-mediated fatty acid oxidation. Cell Death & Disease, 10(3), 158.

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MDSC Inhibition of T Cell Proliferation

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The suppressive function of MDSCs was measured by their ability to inhibit the proliferation of autologous T cells. Fresh T cells were isolated from PBMCs of autologous donors using anti-CD3 microbeads and magnetic LS column separation (Miltenyi Biotech). T cells were then labeled with 1 µM CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) and stimulated with anti-CD3/CD28 mAbs (Invitrogen) and IL-2 (200 U/mL, R&D Systems). Stimulated T cells were seeded at a concentration of 0.5×10⁶ cells/well alone and with autologous MDSCs at 1:1 and 1:2 ratios in a 24-well plate. After 3 days of culture, cells were harvested and stained with CD4-PacBlue (clone 13B8.2) and CD8-APC (clone B9.11) antibodies obtained from BD Biosciences. T cell proliferation was assessed as CFSE dilution determined by flow cytometry.

Nalawade S.A., Shafer P., Bajgain P., McKenna M.K., Ali A., Kelly L., Joubert J., Gottschalk S., Watanabe N., Leen A., Parihar R., Vera Valdes J.F, & Hoyos V. (2021). Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer. Journal for Immunotherapy of Cancer, 9(11), e003237.

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Expansion and Characterization of Human T Cells

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The blood samples were collected from healthy donors (Chinese PLA General Hospital, 2017YFA003003). Then the peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. The isolated human PBMCs were cultured in complete culture media, including advanced RPMI 1640 media (Gibco, 12633012), supplemented with 10% human serum (Sigma, NIST909C), 200U/ml IL2 (MCE, HY-P7039) and 1% penicillin/streptomycin (Pen/Strep, 15140163), at 37°C and 5% CO₂ concentration. MagniSort Human T cell Enrichment Kit (Invitrogen, 8804-6810-74) was used to obtain CD3+ cells. The complete culture media were mixed with magnetic beads coated with anti-CD3/CD28 mAbs (Invitrogen, 11141D) to stimulate and expand the CD3+ T cells. T cells were mixed with magnetic beads at a ratio of 1:1 beads-to-cells. During each induction cycle, 2.5 × 10⁶ cells in a 5 mL/well volume were plated in a 6−well culture plate at 37°C/5% CO₂ for four days. Then magnetic beads were washed away, and cells were cultured in a culture medium for two days. Flow analysis of the T cell exhaustion state was performed after each cycle.

Shi J., Li G., Liu L., Yuan X., Wang Y., Gong M., Li C., Ge X, & Lu S. (2023). Establishment and validation of exhausted CD8+ T cell feature as a prognostic model of HCC. Frontiers in Immunology, 14, 1166052.

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Induction of Regulatory T Cells in CIA Mice

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Naive CD4⁺ T cells were sorted from the splenic cells of mice with CIA, stained with CFSE as described above, and stimulated with an anti-CD3/CD28 mAb (50 ng/ml, PeproTech), IL-1β (10 ng/ml, PeproTech), IL-6 (25 ng/ml, PeproTech), and TGF-β1 (1 ng/ml, PeproTech). Then, iTregs, iTreg_mDC, or iTreg_mtDC were added to the culture at a 1 : 1 ratio. After 4 days of coculture, the cells were harvested, incubated with an anti-mouse CD4-APC mAb, fixed, permeabilized, and stained with an anti-IL-17A-PE mAb (BD Biosciences Pharmingen). The production of IL-17A in the cell culture supernatant was measured using the mouse cytokine cytometric bead array (CBA, BD Biosciences Pharmingen) according to the manufacturer's instructions.

Yang J., Liu L., Yang Y., Kong N., Jiang X., Sun J, & Xie R. (2017). Adoptive Cell Therapy of Induced Regulatory T Cells Expanded by Tolerogenic Dendritic Cells on Murine Autoimmune Arthritis. Journal of Immunology Research, 2017, 7573154.

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Inducible Regulatory T Cell Generation

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iTregs were derived from CD4⁺ CD25⁻ T cells that were purified from the splenocytes of D1 mice using a CD4⁺ CD25⁺ regulatory T cell isolation kit (Miltenyi Biotec, Germany) and stimulated with an anti-CD3/CD28 mAb (50 ng/ml, PeproTech) in the presence of TGF-β1 (5 ng/ml, PeproTech) and IL-2 (100 ng/ml, PeproTech) for 5 days [8 (link)]. Then, iTreg_mtDC or iTreg_mDC were generated by expanding iTregs for 4 days using mtDCs or mDCs at a 5 : 1 ratio (5 T cells : 1 DC). The different types of iTregs were collected, counted, incubated with anti-mouse CD4-FITC and CD25-PE-Cy5 mAbs, fixed, permeabilized, and stained with antimouse Foxp3-PE or isotype control mAbs (BD Biosciences Pharmingen). Then, the coexpression of CD25 and Foxp3 was determined by flow cytometry.

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