Cd3 cd28 activator beads

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

CD3/CD28 activator beads are a specialized lab equipment product designed for T-cell activation. They are magnetic beads coated with antibodies against the CD3 and CD28 receptors on T-cells, which are essential for T-cell activation and proliferation.

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6 protocols using cd3 cd28 activator beads

Glucose-Mediated CD8+ T Cell Activation

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Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors as described elsewhere (20 (link)). Primary human CD8⁺ T cells were negatively isolated from PBMCs using Human CD8⁺ T Cell isolation Kits (Miltenyi Biotec). Human CD8⁺ T cells were stimulated with CD3/CD28 activator beads (Thermo Fisher Scientific) and cultured in DMEM medium (Thermo Fisher Scientific) containing normal (5.6 mM) or high glucose (25 mM) for up to three days if not otherwise mentioned. Levels of glucose in medium was examined every day using “Contour Next Sensoren” test strips (SMS Medipool) and consumed glucose was compensated accordingly. If CTLs were cultured longer than three days, human recombinant IL-2 (Miltenyi Biotec) was added to the medium every two days from day 2 on (100 U/ml). All cells were cultured at 37°C with 5% CO₂. Human pancreatic beta cell line 1.4E7 was purchased from Merck and cultured in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 2 mM glutamine, 1% Penicillin-Streptomycin plus 10% FCS (ThermoFisher Scientific).

Yang W., Denger A., Diener C., Küppers F., Soriano-Baguet L., Schäfer G., Yanamandra A.K., Zhao R., Knörck A., Schwarz E.C., Hart M., Lammert F., Roma L.P., Brenner D., Christidis G., Helms V., Meese E., Hoth M, & Qu B. (2022). Unspecific CTL Killing Is Enhanced by High Glucose via TNF-Related Apoptosis-Inducing Ligand. Frontiers in Immunology, 13, 831680.

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Senescent Cholangiocyte-CD4+ Cell Co-Culture

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Cholangiocytes were seeded at 1 × 10⁵ cells per well in a 24 well companion plate (Corning) in 80 μL phenol red‐free H69 media and cultured overnight. Senescence was induced in cholangiocytes as previously described before addition of CD4+ cells. 0.4 μm inserts (Corning) were placed above with freshly isolated CD4+ cells at a density of 1 × 10⁶ cells in 500 μL Iscove's Modified Dulbecco's Medium (Sigma, Poole, UK) supplemented with 10% FBS, 2 mmol/L L‐glutamine, 100 IU penicillin and 100 μg/mL streptomycin as shown in Figure 1A. CD4+ cells were activated using CD3/CD28 activator beads (Thermo Fisher, Paisley, UK) at a ratio of 1 bead to 2 cells. Co‐cultures were left in culture for 72 hours before analysis.

Etherington R.E., Millar B.J., Innes B.A., Jones D.E., Kirby J.A, & Brain J.G. (2019). Bile acid receptor agonists in primary biliary cholangitis: Regulation of the cholangiocyte secretome and downstream T cell differentiation. FASEB bioAdvances, 1(5), 332-343.

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Diabetes Immune Cell Activation

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Blood samples were collected from patients with diabetes type 1 and 2 (using the current diagnostic criteria from the American Diabetes Association-ADA) and healthy control subjects (all with normal glycated haemoglobin level (HbA1c < 5,7%) at the day of the blood sample collection). Both patients and healthy controls were recruited in the Department of Internal Medicine II in the Saarland University Medical Center, Homburg, Saarland, Germany. Written informed consent was obtained from all subjects before blood sampling, strictly following the procedure described in the ethical approval of the ethic committee of the medical association of Saarland (Ethic Vote Nr: Ha 84/19). PBMCs were obtained from the blood samples and were stimulated with CD3/CD28 activator beads (Thermo Fisher Scientific) and cultured in DMEM containing normal (5.6 mM) or high glucose (25 mM) for three days, supplemented with recombinant IL-2 (100 U/ml, Miltenyi).

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Culturing CD8+ T Cells under Glucose Conditions

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Raji cells were cultured in RPMI-1640 medium (ThermoFisher Scientific) containing 10% FCS and 1% Penicillin-Streptomycin. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors as described before (24 (link)). Primary human CD8⁺ T cells were negatively isolated from PBMCs using Human CD8⁺ T Cell isolation Kits (Miltenyi Biotec) according to the manufacturer’s instruction. Human CD8⁺ T cells were stimulated with CD3/CD28 activator beads (ThermoFisher Scientific) and cultured in DMEM containing normal (5.6 mM, NG) or high glucose (25 mM, HG) (ThermoFisher Scientific) for 3 days if not otherwise mentioned. Since day 2, additional glucose was added into the medium every two days to compensate the consumed glucose along with recombinant human IL-2 (50 ng/ml, ThermoFisher Scientific). All cells were cultured at 37°C with 5% CO₂. EG7 mouse lymphoma cell line was stably transfected with 4 µg of pCasper-pMax plasmid (25 (link)) using SF Cell Line 4D-NucleofectorTM X Kit (Lonza). 48h after transfection, cells were treated with 0.4 mg/ml G418 and 4 µg/ml puromycin in RPMI-1640 (ThermoFisher Scientific) with 10% FCS. Monoclonally expanded EG7-pCasper cells were cultured in selection medium, supplemented with 1% Penicillin/Streptomycin. Flow cytometry analysis revealed 98% of EG7-pCasper cells were fluorescent.

Zhu J., Yang W., Zhou X., Zöphel D., Soriano-Baguet L., Dolgener D., Carlein C., Hof C., Zhao R., Ye S., Schwarz E.C., Brenner D., Prates Roma L, & Qu B. (2021). High Glucose Enhances Cytotoxic T Lymphocyte-Mediated Cytotoxicity. Frontiers in Immunology, 12, 689337.

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Expanded AAV-edited Cell Sorting

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AAV edited cells were expanded for seven days in culture then rested 24 hours without cytokine. Cells were then bulk sorted based on editing outcome, using a FACS Aria I. Cells were gated on size and singlets, then sorted on BFP/GFP positivity. After sorting cells were expanded with CD3/CD28 Activator Beads (Gibco) at 1 bead to 50 cells (ratios of beads to cells higher than 1:25 caused severe activation induced cell death).

Anderson W., Thorpe J., Long S.A, & Rawlings D.J. (2019). Efficient CRISPR/Cas9 disruption of autoimmune-associated genes reveals key signaling programs in primary human T cells. Journal of immunology (Baltimore, Md. : 1950), 203(12), 3166-3178.

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Comprehensive Immunophenotyping of T Cells

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Anti-human CD4FITC (eBioscience 11-0049)), CD3PerCpCy5.5 (eBioscience 45-0037), CD8PE (eBioscience 12-0088), CD45ROFITC, CD45RAPE, TNFα eFlour450 (eBioscience 48-7349), IFNγAPC (eBioscience 17-7319), TbetPE (eBioscences 12-5825-80), eomesPE-eFlour610 (eBioscience 61-4877) and appropriate isotype control antibodies as well as human Fc Receptor binding inhibitor (eBioscience 14-9161), Protein transport inhibitor (Brefeldin A and Monensin (eBioscence 00-4980)), PMA and Ionomycin (T cell stimulation cocktail (eBioscience 00-4970 )), Intracellular fixation and permeabilization kit (eBioscience 88-8824) FOXP3/transcription factor staining kit (eBioscience 00-5523). FACS lysing buffer was obtained from BD Biosciences (BD 349202). CD279 PECy7 (BioLegend 329917), CD8AF700 (BioLegend 301028), CCR7APC (BioLegend 353214), GranzymeBAlexaFlour647 (BioLegend 515405), CD107eFlour450 (eBioscience 48-1079) and appropriate isotype controls. CD3/CD28 activator beads (Gibco 11131D), EBV peptides; HLA A2.1 (GLCTLVAML), HLA A2.2 (CLGGLLTMV), HLA A2.3 (LLDFVRFMGV), HLA B7.1 (RPPIFIRRL), HLA B7.2 (VPAPAGPIV), HLA B8.1 (RAKFKQLL), HLA B8.2 (QAKWRLQTL), HLA B8.3 (FLRGRAYGL). Peptides were obtained from Invitrogen.

Sponaas A.M., Yang R., Rustad E.H., Standal T., Thoresen A.S., Dao Vo C., Waage A., Slørdahl T.S., Børset M, & Sundan A. (2018). PD1 is expressed on exhausted T cells as well as virus specific memory CD8+ T cells in the bone marrow of myeloma patients. Oncotarget, 9(62), 32024-32035.

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