Dynabeads human t cell activator cd3cd28

Manufactured by Thermo Fisher Scientific

Sourced in Ireland, United States, United Kingdom

Dynabeads Human T Cell Activator CD3/CD28 is a magnetic bead-based product designed to activate and expand human T cells in vitro. It contains antibodies targeting the CD3 and CD28 surface markers on T cells, which are essential for their activation and proliferation.

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Lab products found in correlation

Human duoset elisa kit, by R&D Systems (2 mentions) Opteia human elisa kit, by BD (2 mentions) Celltrace violet, by Thermo Fisher Scientific (2 mentions) Celltrace cfse, by Thermo Fisher Scientific (2 mentions) Human recombinant il 23, by BioLegend (2 mentions) Soluble anti cd28 mab, by BioLegend (2 mentions) Human recombinant tgfβ1, by BioLegend (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Knockout serum replacement (ksr), by Thermo Fisher Scientific (2 mentions) Sil2rα, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) 2 β mercaptoethanol, by Merck Group (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) Human serum, by Euroclone (1 mentions) Crl 3216, by American Type Culture Collection (1 mentions) X vivo, by Lonza (1 mentions) Cfse kit, by Thermo Fisher Scientific (1 mentions) Human factor 5 elisa kit, by Abcam (1 mentions) Miltenyi isolation kits, by Miltenyi Biotec (1 mentions) Cellfix, by BD (1 mentions)

24 protocols using dynabeads human t cell activator cd3cd28

CD4+ T Cell Proliferation and Suppression

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The proliferation assay was performed as previously described ¹¹ CD4⁺ T cells labelled by CellTrace CFSE (Life Technologies, Carlsbad, CA, USA) were activated by Dynabeads Human T‐cell Activator CD3/CD28 at 1:25 bead:cell ratio. After 96 hours of stimulation, the proliferation of CD4⁺ T cells was counted by FACS.
The suppression assay was performed as previously described.¹¹ Briefly, responder CD4⁺ T cells (50 000 cells) labelled by CFSE and suppressor CD4⁺ T cells (12 500–50 000 cells) labelled by CellTrace Violet (Life Technologies) were co‐cultured at different concentrations (1:0.25–1:1) after activation by Dynabeads Human T‐cell Activator CD3/CD28 at 1:25 bead:cell ratio. After 96 hours of stimulation, the proliferation of responder CD4⁺ T cells was counted by FACS.

Sato Y., Passerini L., Piening B.D., Uyeda M.J., Goodwin M., Gregori S., Snyder M.P., Bertaina A., Roncarolo M, & Bacchetta R. (2020). Human‐engineered Treg‐like cells suppress FOXP3‐deficient T cells but preserve adaptive immune responses in vivo. Clinical & Translational Immunology, 9(11), e1214.

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T Cell Suppression Assay Protocol

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Suppression assay was performed as previously described [23 (link),24 ]. Briefly, responder CD4⁺ T cells (50,000 cells) labeled with Cellstain CFSE (FUJIFILM/Wako Pure Chemical) and suppressor CD4⁺ T cells (12,500–50,000 cells) labeled with Cellstain CytoRed (FUJIFILM/Wako Pure Chemical) were re-suspended in 100

μ

l of ImmunoCult XF T cell expansion medium without rhIL-2. Responder and suppressor cells were co-cultured at different concentrations (1:0.25–1:1) after activation by Dynabeads Human T cell Activator CD3/CD28 (Gibco) at a 1:25 bead-to-cell ratio in 96 well plates. After 96 h of stimulation, proliferation of responder CD4⁺ T cells was determined by Flow Cytometry. The suppression index was calculated as previously described [25 (link)].

Sato Y., Osada E, & Manome Y. (2023). Non-canonical NFKB signaling endows suppressive function through FOXP3-dependent regulatory T cell program. Heliyon, 9(12), e22911.

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Isolation and Differentiation of CD8+ T Cells

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Samples of 5 ml blood were collected from healthy human volunteers, and PBMCs were isolated through Ficoll-dependent density gradient centrifugation. CD8⁺ T cells were then isolated from PBMCs by negative selection using Dyna beads (Invitrogen, MA, USA) according to the manufacturer’s protocol. Cell purity was checked by flow cytometry and was ascertained to be around 85~90% consistently (Supplementary Material Figure S2a,b). Isolated cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum of Australian origin (PAN-Biotech, Aidenbach, Germany), 100 U/mL penicillin, 100 μg/mL streptomycin and 50 mM 2β- mercaptoethanol (Sigma, Burlington, MA, USA). For Tc1 differentiation, 1 × 10⁶ CD8⁺ T cells were activated by Dyna beads human T cell activator CD3/CD28 (Gibco, Dublin, Ireland) according to the manufacturer’s protocol. Additionally, IL-12 (10 ng/mL), IL-2 (100 IU/mL) and anti-IL-4 (10 mg/mL) was added after 24 h. For Tc21 differentiation, IL-21(25 ng/mL) was added along with the above conditions. After 10 days of activation, the cells were then washed with RPMI 1640 media and used for subsequent experiments as indicated [27 (link),28 (link)].

Sengupta S., Bhattacharya G., Mohanty S., Shaw S.K., Jogdand G.M., Jha R., Barik P.K., Parida J.R, & Devadas S. (2023). IL-21, Inflammatory Cytokines and Hyperpolarized CD8+ T Cells Are Central Players in Lupus Immune Pathology. Antioxidants, 12(1), 181.

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FOXP3KO T Cell Cytokine Profiling

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Transduced FOXP3KO^GFP CD4+ T cells were cultured at the density of 1 × 10⁶ cells/ml in a round-bottom 96-well plate and activated by Dynabeads Human T-cell Activator CD3/CD28 (Gibco) at a 1:25 bead: cell ratio. Culture supernatant was collected at 24 h (IL-2) and 72 h (IL-4, IFN-γ, IL-17A, IL-22) after stimulation. Cytokine concentrations, including IL-2, IL-4, and IFN-γ; were measured by Human OptEIA ELISA Kit (BD Biosciences). Cytokine concentrations, including IL-17A and IL-22 concentrations, were measured by Human DuoSet ELISA Kit (R&D).

Sato Y., Liu J., Lee E., Perriman R., Roncarolo M.G, & Bacchetta R. (2021). Co-Expression of FOXP3FL and FOXP3Δ2 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function. Frontiers in Immunology, 12, 752394.

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Suppression Assay for FOXP3KO CD4+ T Cells

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Suppression assay for transduced FOXP3KO^GFP CD4+ T cells was done according to the previous publication (18 (link)). Briefly, responder CD4⁺ T cells (50,000 cells) labeled by CellTrace CFSE (Life Technologies) and transduced FOXP3KO^GFP suppressor cells (12,500–50,000 cells) labeled by CellTrace Violet (Life Technologies) were cocultured at different concentrations (1:0.25–1:1) after activation by Dynabeads Human T-cell Activator CD3/CD28 (Gibco) at the 1:25 bead: cell ratio. Ninety-six hours after stimulation, proliferations of responder CD4⁺ T cells were counted by FACS.

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T-cell Activation and Cytokine Profiling

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CD4⁺ T cells were cultured at a density of one million of cells mL⁻¹ in a round‐bottom 96‐well plate and activated using the Dynabeads Human T‐cell Activator CD3/CD28 (Gibco, Gaithersburg, MD) at a 1:25 bead:cell ratio. Culture supernatant was collected at 24 h (IL‐2) and 72 h (IL‐4, IFN‐γ, IL‐17A, IL‐22) after stimulation. Cytokine concentrations including IL‐2, IL‐4 and IFN‐γ were measured by Human OptEIA ELISA Kit (BD Bioscience). Cytokine concentrations including IL‐17A and IL‐22 concentrations were measured by Human DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA).

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Lentiviral Vector-Mediated Gene Transduction of T-Cells

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The bidirectional SIN.LV.PGK.BACH2 and SIN.LV.PGK.STAT5B plasmid constructs were generated using Gateway Technology (for details refer to Supplementary Methods). All concentrated VSV.G-pseudotyped LV stocks were produced by transient transfection of 293 T cells (CRL-3216, ATCC), as previously reported^{7 (link)}.
For transduction, Naïve T lymphocytes were pre-activated for 24 h in X-VIVO (Lonza), 5% human serum (Euroclone), in presence of anti-CD3/CD28 activating beads (Dynabeads Human T cell activator CD3/CD28, Life Technologies). Treg and Teff cells were pre-activated for 24 h in X-VIVO (Lonza), 5% human serum (Euroclone), in presence of Human Treg Expander beads (Life Technologies). Cells were infected with the indicated LV at MOI 20.

Cesana D., Santoni de Sio F.R., Rudilosso L., Gallina P., Calabria A., Beretta S., Merelli I., Bruzzesi E., Passerini L., Nozza S., Vicenzi E., Poli G., Gregori S., Tambussi G, & Montini E. (2017). HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells. Nature Communications, 8, 498.

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In vitro generation of human Tfh cells

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Human Tfh cells were generated as previously described^{42 (link)}. Briefly, after overnight stimulation of naive CD4⁺ T cells with Dynabeads human T cell activator CD3/CD28 (Thermo Fisher) in complete RPMI medium, cells were transferred to 96-well plates coated with anti-CD3 and supplemented with 2 μg/ml of soluble anti-CD28 mAb (Biolegend), human recombinant IL-23 (25 ng/ml; Biolegend) and human recombinant TGFβ1 (5 ng/ml; Biolegend). After 4 d cells were collected and used for analysis.

Caielli S., Troggian Veiga D., Balasubramanian P., Athale S., Domic B., Murat E., Banchereau R., Xu Z., Chandra M., Chung C.H., Walters L., Baisch J., Wright T., Punaro M., Nassi L., Stewart K., Fuller J., Ucar D., Ueno H., Zhou J., Banchereau J, & Pascual V. (2018). A CD4+ T cell population expanded in Lupus blood provides B cell help through IL10 and succinate. Nature medicine, 25(1), 75-81.

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In vitro generation of human Tfh cells

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Modulation of T Cell Proliferation by Factor V

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B cell proliferation kit (R&D system, Abingdon, UK). BD CellFix (BD Biosciences, Oxford, UK). Native human FV, FVa, Thrombin, Dynabeads Human T-cell activator, and CFSE kits (Thermofisher Scientific, Loughborough, UK). Human Factor V ELISA Kit (ab137976, Abcam, Cambridge, UK). Unless otherwise indicated, all reagents were from Sigma-Aldrich Company Ltd (Dorset, UK).
CD4 T cell, CD8 T cell and B cell were isolated using Miltenyi isolation kits (Miltenyi Biotec, Surrey, UK) following the manufacturer’s instruction. Cells were stained with CFSE using CFSE kits (Thermofisher Scientific, Loughborough, UK) following the manufacturer’s instruction. 5 × 10⁴ labelled T cons were mixed with Dynabeads Human T cell activator CD3/CD28 (Thermofisher Scientific, Loughborough, UK) at cell-to-beads ratio of 2 to 1 and incubated at 37°C. Cells incubated with native human FV, FVa, FV constructs at the concentrations indicated, thrombin (5 μg/mL), and hirudin (5 iU/ml) were collected and analysed using a BD Fortessa flow cytometer after 4 or 5 days.

Wang J., Kotagiri P., Lyons P.A., Al-Lamki R.S., Mescia F., Bergamaschi L., Turner L., Morgan M.D., Calero-Nieto F.J., Bach K., Mende N., Wilson N.K., Watts E.R., Maxwell P.H., Chinnery P.F., Kingston N., Papadia S., Stirrups K.E., Walker N., Gupta R.K., Menon D.K., Allinson K., Aitken S.J., Toshner M., Weekes M.P., Nathan J.A., Walmsley S.R., Ouwehand W.H., Kasanicki M., Göttgens B., Marioni J.C., Smith K.G., Pober J.S, & Bradley J.R. (2022). Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19. iScience, 25(3), 103971.

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