αcd3 αcd28 dynabeads

Manufactured by Thermo Fisher Scientific

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αCD3/αCD28 Dynabeads are paramagnetic beads coated with antibodies against CD3 and CD28 proteins. They are used for the activation and expansion of T cells in cell culture applications.

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Lab products found in correlation

Celltrace violet, by Thermo Fisher Scientific (2 mentions) Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions) Facsverse, by BD (1 mentions) Interleukin 2 (il 2), by Merck Group (1 mentions) Macsquant analyzer 10, by Miltenyi Biotec (1 mentions) Interleukin 6 (il 6), by CellGenix (1 mentions) Cytofix cytoperm fixation permeabilization, by BD (1 mentions) Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions) Anti ifny, by BD (1 mentions) Negative selection kit, by Thermo Fisher Scientific (1 mentions) Pan t cell isolation kit, by Miltenyi Biotec (1 mentions) Macs ms column, by Miltenyi Biotec (1 mentions) Facsaria 2, by BD (1 mentions) Murine il 2, by Thermo Fisher Scientific (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions) Na ve t cell isolation kit, by STEMCELL (1 mentions) Cd4 cell excluding magnetic beads, by Miltenyi Biotec (1 mentions) X vivo 15 media, by Lonza (1 mentions)

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Dynabeads (2112 citations)

10 protocols using αcd3 αcd28 dynabeads

T Cell Proliferation and Activation in 2D and 3D Environments

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Human pan T cells were stained with CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) to track cell proliferation by flowcytometry. To pre-activate T cells, pan T cells were stimulated overnight with αCD3/αCD28 Dynabeads (Gibco). The following day, T cells were harvested and re-plated into two-dimensional (2D) medium, 3D collagen matrices or 3D RGD-PIC hydrogels at varying cell densities as indicated. Alternatively, unstimulated T cells (1 × 10⁶ cells/ml) were mixed with αCD3/αCD28 Dynabeads (Gibco) or with IL-2 (90-100 IU/mL), PHA (phytohaemagglutinin, 1 μg/mL, Sigma Aldrich) and IL-6 (15 ng/mL, Cell Genix). Subsequently, cells were embedded within the RGD-PIC hydrogels or collagen matrices at varying densities to test in situ activation. Cells were recovered from the RGD-PIC gels and collagen gels and proliferation by CFSE dilution and activation were assessed by flowcytometry on a FACS Verse (BD Biosciences). Cell numbers were quantified using a MACSQuant Analyzer 10 (Miltenyi Biotec). Fixable Viability Dye eFluor® 780 (eBioscience) was used to exclude dead cells. Intracellular staining for interferon gamma (IFNy) was done using anti-IFNy (BD Biosciences) and the BD Cytofix/Cytoperm Fixation/Permeabilization.

Weiden J., Voerman D., Dölen Y., Das R.K., van Duffelen A., Hammink R., Eggermont L.J., Rowan A.E., Tel J, & Figdor C.G. (2018). Injectable Biomimetic Hydrogels as Tools for Efficient T Cell Expansion and Delivery. Frontiers in Immunology, 9, 2798.

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T Cell Isolation and Activation

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Human peripheral blood mononuclear Cells (PBMCs) were isolated from leukopheresis by Ficoll-Hypaque density gradient. Isolated human PBMCs were subjected for CD3+ T cell isolation by using the Pan T cell Isolation Kit (Miltenyi) as instructed in the manual. The isolated cells were activated for 72 hr with αCD3/αCD28 Dynabeads (Invitrogen) at a cell to bead ratio of 1:1 in RPMI+10% Fetal Bovine Serum (supplemented with HEPES buffer, Penicillin/Streptomycin, and L-glutamine). Murine CD4+ and CD8+ T cells were isolated from the spleen and lymph nodes using the Negative Selection Kit (Invitrogen), and were activated with plate-coated αCD3 (10μg/ml) and soluble αCD28 (2μg/ml) or with cognate Ova peptides in the presence irradiated splenocytes for 72 hr.

Park B.V., Freeman Z.T., Ghasemzadeh A., Chattergoon M.A., Rutebemberwa A., Steigner J., Winter M.E., Huynh T.V., Sebald S.M., Lee S.J., Pan F., Pardoll D.M, & Cox A.L. (2016). TGF-β1-mediated Smad3 enhances PD-1 expression on antigen-specific T cells in cancer. Cancer discovery, 6(12), 1366-1381.

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Isolation and Activation of T Cells

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Blood bags from anonymous healthy donors were obtained from the Welsh Blood Service (Pontyclun, UK). Lymphocytes were purified using lymphoprep (Axia‐Shield, Dundee, UK) and typed for HLA A2 by antibody staining. CD8⁺ and CD4⁺ T cells were selected positively by CD8 and CD4 microbeads, respectively, purified through a magnetic affinity cell sorting (MACS) MS column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended at 10⁶/well in R10 with IL‐15, IL‐2 and Cellkines. Cells were activated overnight with αCD3/αCD28 Dynabeads (Invitrogen) at a bead to cell ratio of 3:1 before lentiviral transduction.

Tan M.P., Dolton G.M., Gerry A.B., Brewer J.E., Bennett A.D., Pumphrey N.J., Jakobsen B.K, & Sewell A.K. (2016). Human leucocyte antigen class I‐redirected anti‐tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells. Clinical and Experimental Immunology, 187(1), 124-137.

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Suppressive Capacity of Tumor-Infiltrating Tregs

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FIR mice were challenged with ID8agg, treated with IL-2c or isotype and sacrificed 5 weeks post tumor challenge. CD45⁺CD3⁺CD4⁺CD25^hiRFP⁺ Tregs were sorted by flow cytometry (95–99% purity) from TDLN and ascites and incubated with 30,000 CFSE-stained, splenic CD45⁺CD3⁺CD4⁺CD25⁻ responder T cells (95–99% purity) obtained by electronic cell sorting from naïve, aged matched syngeneic females. T cells from naïve WT mice, at graded concentrations plus αCD3/αCD28 Dynabeads (Invitrogen; 1 bead for every 5 effector cells) in a round-bottom 96-well plate for ~80 hours. Proliferation was analyzed by flow cytometry based on the dilution of CFSE.

Drerup J.M., Deng Y., Pandeswara S.L., Padrón Á.S., Reyes R.M., Zhang X., Mendez J., Liu A., Clark C.A., Chen W., Conejo-Garcia J.R., Hurez V., Gupta H, & Curiel T.J. (2020). CD122-selective IL-2 complexes reduce immunosuppression, promote Treg fragility, and sensitize tumor response to PD-L1 blockade. Cancer research, 80(22), 5063-5075.

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T Cell Activation with Monocyte-Derived EVs

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CFSE-stained T cells were seeded on anti-CD3 coated 96-well plates or stimulated T cells were plated at 1 × 10⁵ cells per well with αCD3/αCD28 DynaBeads (Thermo Fisher). Monocytes previously conditioned with EVs derived from untreated or IFN-γ-stimulated GBM cells were resuspended in serum-free DMEM/F12 and added to T cells in a 3:1 monocyte:T cell ratio. After 5 days of incubation, T cells were harvested and analyzed by flow cytometry as per above.

Jung M.Y., Aibaidula A., Brown D.A., Himes B.T., Cumba Garcia L.M, & Parney I.F. (2022). Superinduction of immunosuppressive glioblastoma extracellular vesicles by IFN-γ through PD-L1 and IDO1. Neuro-Oncology Advances, 4(1), vdac017.

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G-MDSC Suppression of T Cell Proliferation

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Single-cell suspension of 6 tumors from each group (ATRA-treated and untreated) were pooled 10 days after tumor inoculation. Live G-MDSCs (MHCII^loCD11b⁺Ly6C^loLy6G⁺) were sorted with BD FACSAria II (BD Biosciences). Splenic CD3⁺ T cells were isolated from naïve mice utilizing magnetic negative selection (BioLegend). T cells were labeled with CellTrace Violet (Invitrogen) and plated in a 96-well U-bottom plate (1×10⁵ cells per well) in complete media. Flow-sorted G-MDSCs from control or ATRA-treated tumor-bearing mice were added at varying proportions to T cells and incubated overnight. The following day, T cells were treated with 1×10⁵ αCD3/αCD28 Dynabeads (ThermoFisher) and 10ng/mL murine IL2 (PeproTech). T cell proliferation was evaluated by flow cytometry 4 days after stimulation. For in vitro treatment, sorted G-MDSCs from control mice were pre-treated with 2μM ATRA for 24 hours. Culture media was replaced with fresh media to remove ATRA prior to co-culturing with T cells.

Li R., Salehi-Rad R., Crosson W., Momcilovic M., Lim R.J., Ong S.L., Huang Z.L., Zhang T., Abascal J., Dumitras C., Jing Z., Park S.J., Krysan K., Shackelford D.B., Tran L.M., Liu B, & Dubinett S.M. (2021). Inhibition of Granulocytic Myeloid-Derived Suppressor Cells Overcomes Resistance to Immune Checkpoint Inhibition in LKB1-deficient Non-Small Cell Lung Cancer. Cancer research, 81(12), 3295-3308.

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PBMC Isolation, Fatty Acid Stimulation, and Antioxidant Treatment

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Fifty milliliters of peripheral blood were collected into acid/citrate/dextrose containing tubes by venous puncture. PBMCs were purified by histopaque 1077 then frozen at −80 °C under controlled cooling conditions in a Mr. Frosty apparatus (Nalgene, Sigma Aldrich, St Louis, MO, USA). For multi-week storage, cells were moved to −170 °C following 1–7 days at −80 °C. PBMCs from the subjects were stimulated in vitro for 40 h with T cell-targeted αCD3/αCD28 Dynabeads (Thermo Fisher Scientific, 11132D, Waltham, MA, USA) at 2 μL Dynabeads per 100k cells. In some cultures, cells were co-treated with 400 µM palmitate (pal) (C16:0) coupled to fatty acid-free Bovine Serum Albumin (BSA) at a ratio of 2 mol palmitate to 1 mole BSA, or 400 µM oleate, or a combination of palmitate and oleate. These fatty acid concentrations mimic concentrations achievable in serum [13 (link)]. Control cells were treated with 1% BSA. The mitochondrial ROS scavenger MitoTempo (mito)(10 μM) or a general ROS scavenger N-acetyl cysteine (NAC) was added for the last 20 h of incubation (20 h post-stimulation) for some cultures. All treatments were done in RPMI media with 5 mM glucose (normoglycemic). Supernatants were collected and stored at −80 °C. Cells were assayed as outlined below.

McCambridge G., Agrawal M., Keady A., Kern P.A., Hasturk H., Nikolajczyk B.S, & Bharath L.P. (2019). Saturated Fatty Acid Activates T Cell Inflammation Through a Nicotinamide Nucleotide Transhydrogenase (NNT)-Dependent Mechanism. Biomolecules, 9(2), 79.

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Modulation of Naive T Cell Proliferation by Treg and Cytokines

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Naïve T cells from spleen and mLN were enriched from Il23r^flox/flox;Cd2^Cre mice (lacking IL-23R in all lymphocytes) using a naïve T cell isolation kit (Stem Cell Technologies) and labeled with CellTrace Violet (Thermo Fisher Scientific) according to manufacturer’s protocol. Cells were counted and stimulation was performed using αCD3αCD28 Dynabeads (Thermo Fisher). Cells were cultured for 72 hours together with 10,000 FACS-sorted WT colonic YFP⁺ Treg cells in a 1:1 Treg:Tnaïve ratio in the presence or absence of 20 ngmL⁻¹ recombinant mIL-23 or IL-6 (R&D Systems). Proliferation was assessed via CellTrace Violet dilution in naïve T cells and % suppression determined.

Jacobse J., Brown R.E., Li J., Pilat J.M., Pham L., Short S.P., Peek C.T., Rolong A., Washington M.K., Martinez-Barricarte R., Byndloss M.X., Shelton C., Markle J.G., Latour Y.L., Allaman M.M., Cassat J.E., Wilson K.T., Choksi Y.A., Williams C.S., Lau K.S., Flynn C.R., Casanova J.L., Rings E.H., Samsom J.N, & Goettel J.A. (2023). Interleukin-23 receptor signaling impairs the stability and function of colonic regulatory T cells. Cell reports, 42(2), 112128.

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Metabolic Profiling of Aged CD4+ T Cells

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Fifty milliliters of peripheral blood were collected into acid/citrate/dextrose containing tubes by venous puncture. PBMCs were purified by histopaque 1077 followed by negative selection with CD4⁺ cell-excluding magnetic beads (Miltenyi, Auburn, CA) for experiments on purified T cells as we published ⁴. CD4⁺ T cells were >93% pure as assessed via flow cytometry. All cells were frozen at -80°C in a Mr. Frosty apparatus (Nalgene). For multi-week storage, cells were moved to -170°C following 1-7 days at -80°C. T cells from BMI-matched young (Y) and old (O) subjects were stimulated in vitro for 40 h with αCD3/αCD28 Dynabeads (Thermo Fisher Scientific, 11132D) at 2μL/100k cells ± 100μM metformin (met) in RPMI media with 5mM glucose (euglycemic). The supernatant was stored at -80°C until analyzed in technical duplicate on a BioRad 3D instrument using a Th17 multiplex kit (Miltenyi). Cells were assayed as outlined below. Metabolic characterization of CD4⁺ T cells was performed using the Seahorse XF96 Analyzer (Nicholas et al., 2017 (link), van der Windt et al., 2016 ).

Bharath L.P., Agrawal M., McCambridge G., Nicholas D.A., Hasturk H., Liu J., Jiang K., Liu R., Guo Z., Deeney J., Apovian C.M., Snyder-Cappione J., Hawk G.S., Fleeman R.M., Pihl R.M., Thompson K., Belkina A.C., Cui L., Proctor E.A., Kern P.A, & Nikolajczyk B.S. (2020). Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation. Cell Metabolism, 32(1), 44-55.e6.

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Manufacturing of CMV-Specific TCR-T Cells

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CMV-TCR-T cells were manufactured by Beijing Yongtai Ruike Biotechnology Company. T cells were isolated from the PBMCs of SCT donors and activated by the α-CD3/α-CD28 Dynabeads (Gibco 40203D) for 2 days. Activated T cells were transduced with pLV4 lentiviral vector containing the encoding genes of CMV-specific TCR. Both CD4⁺ and CD8⁺ T cells were transduced. After lentiviral transduction, the T cells were cultured in X-VIVO 15 media (Lonza 02-060Q) supplemented with 200 IU/mL IL-2 at 37°C and 5% CO₂ for approximately 5–11 days to obtain sufficient cells and then cryopreserved. The number of CMV-TCR-T cells manufactured was at least twofold of the maximum required doses of a certain patient before cryopreservation to ensure this patient get sufficient number of CMV-TCR-T cells for infusion after thawing. Cell staining with peptide–major histocompatibility complex (MHC) tetramer and CD3/CD4/CD8 antibodies and flow cytometry analysis were performed to determine the transduction efficiency.

Ma C., Chen P., Du J., Wang L., Lu N., Sun J., Qilong X., Wang Y., Dou L, & Liu D.H. (2024). Adoptive transfer of CMV-specific TCR-T cells for the treatment of CMV infection after haploidentical hematopoietic stem cell transplantation. Journal for Immunotherapy of Cancer, 12(1), e007735.

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