Anti cd28

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom

Anti-CD28 is a laboratory reagent used in immunology research. It functions as an antibody that binds to the CD28 receptor on the surface of T cells, providing a co-stimulatory signal that enhances T cell activation and proliferation.

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Anti-CD3 and anti-CD28 (19 citations) Anti-mouse CD28 (clone 37.51, (7 citations) Soluble anti-CD28 mAb (6 citations) Anti-mouse CD3/CD28 Dynabeads (3 citations) Anti-CD3/CD28 monoclonal antibodies (2 citations) Anti-CD3/CD28 conjugated magnetic beads (2 citations) Anti-CD3/CD28-coated paramagnetic beads (2 citations) Anti-human CD28 (Functional grade purified, (2 citations) Mouse anti-human CD28 (Clone 28.2; (2 citations) Anti-hCD3/CD28-conjugated Dynabeads (2 citations)

306 protocols using anti cd28

CFSE-labeled Tfh Cells Suppression by MDSC

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CD4⁺CXCR5⁺ Tfh cells were sorted by flow cytometry from normal C57BL/6 mice splenocytes. CD11b⁺Gr-1⁺ MDSC were sorted from S. japonicum-infected C57BL/6 mice splenocytes by the Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotech, USA).
Freshly prepared Tfh cells were incubated with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (final concentration of 2 μM; Dojindo Laboratory, Japan) for 15 min at 37 °C. Cells were then resuspended and washed in PBS containing 10% fetal bovine serum. CFSE-labelled Tfh cells were divided into three groups: unstimulated, stimulated with anti-CD3 (5 μg/ml; PeproTech, USA) and anti-CD28 (2 μg/ml, PeproTech), stimulated with anti-CD3 and anti-CD28 plus MDSC. Cells were harvested to analyse the CFSE fluorescence intensity of Tfh by flow cytometry after 5 days.

Zhang Y., Wu Y., Liu H., Gong W., Hu Y., Shen Y, & Cao J. (2021). Granulocytic myeloid-derived suppressor cells inhibit T follicular helper cells during experimental Schistosoma japonicum infection. Parasites & Vectors, 14, 497.

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Measuring Th17 and CD4+ T Cell Cytokines

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To detect IL-17 produced by Th17 cells, cells were stimulated with 1 μg/mL anti-CD3 (eBioscience) and 1 μg/mL anti-CD28 (eBioscience) for 24 h. To detect other cytokines produced by the CD4⁺ T cells, cells were cultured with 1 μg/mL anti-CD3 and 1 μg/mL anti-CD28 for 8 h or 24 h. Culture supernatants were collected at the relevant times. To measure the concentration of cytokines in culture supernatants we used ELISA kits for IFN-γ (eBioscience) and IL-17 (R&D Systems) according to the manufacturers’ instructions. We analyzed the cytokine concentrations using microplate reader software (Thermo Fisher Scientific).

Choi S., Park H., Jung S., Kim E.K., Cho M.L., Min J.K., Moon S.J., Lee S.M., Cho J.H., Lee D.H, & Nam J.H. (2017). Therapeutic Effect of Exogenous Truncated IK Protein in Inflammatory Arthritis. International Journal of Molecular Sciences, 18(9), 1976.

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Neutrophil-T Cell Coculture Assay

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Before coculture with T cells, neutrophils were washed three times with PBS. In a 3-day incubation, bead-purified peripheral CD3⁺ T cells (1 × 10⁵ cells/well in 96-well plates) were labeled with carboxy fluorescein succinimidyl ester (CFSE, Stem Cell Technology) and cocultured with neutrophils at a 1:1 ratio in 100 μl RPMI-1640 medium containing anti-CD3 (8 μg/ml) and anti-CD28 (5 μg/ml) antibodies (eBioscience). In a 1-day coculture system, neutrophils were cocultured with bead-purified CD3⁺ T cells at a 1:1 ratio in 100 μl RPMI-1640 medium containing anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) antibodies (eBioscience). After 1-day incubation, the cells were harvested for flow cytometric analysis of CD69 and interferon (IFN)-γ. For blockade study, the treated neutrophils were cocultured with bead-purified CD3⁺ T cells in the presence of a neutralizing antibody against human PD-L1 (2 μg/ml) (R&D Systems).

Shi Y., Zhang J., Mao Z., Jiang H., Liu W., Shi H., Ji R., Xu W., Qian H, & Zhang X. (2020). Extracellular Vesicles From Gastric Cancer Cells Induce PD-L1 Expression on Neutrophils to Suppress T-Cell Immunity. Frontiers in Oncology, 10, 629.

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T Cell Proliferation Assay with PMN-MDSCs

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Naïve splenocytes were labeled with 2.5 μM CFSE (Invitrogen Molecular Probes) and cultured in RPMI-1640 supplemented with 10% FBS, 20 U/mL recombinant IL-2 (Thermo Fisher Scientific), 1 μg/mL anti-CD3 (Thermo Fisher Scientific), and 5 μg/mL anti-CD28 (Thermo Fisher Scientific). Splenocytes were cultured alone or with freshly sorted PMN-MDSCs at the indicated ratio for 84 h at 37°C in a 5% CO₂ humidified atmosphere. Subsequently, CFSE dilution was assessed using a Cytoflex S flow cytometer (Beckman Coulter); splenocyte number was determined using the FlowJo Software (version 13; Tree Star).

Tao H., Liu M., Wang Y., Luo S., Xu Y., Ye B., Zheng L., Meng K, & Li L. (2021). Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation. Frontiers in Immunology, 12, 609295.

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Activation and Expansion of Murine T Cells

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Lymph Nodes were harvested from the mice and a single cell suspension in PBS made by passing the tissue through a 10 μm strainer. The cells were counted and seeded at density of 10⁶ cells/ml in RPMI supplemented with10% heat inactivated FBS (Labtech), 2 mM L-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin and 50 μM β-mercaptoethanol and stimulated with 1 μg/ml anti-CD28 (cat:16-0281-82; clone:37.51 Thermo Fisher), 1 μg/ml anti CD3e (cat:16-0031-85, Clone:145-2C11 Thermo Fisher), 20 ng/ml of IL-2 and 1 ng/ml of IL-12. The cells were cultured for 36 h and then the cell suspension was diluted 5 times with RPMI media containing 20 ng/ml of IL-2. The cells were seeded and maintained at 300,000 cells/ml and split every consequent day.

Remenyi J., Naik R.J., Wang J., Razsolkov M., Verano A., Cai Q., Tan L., Toth R., Raggett S., Baillie C., Traynor R., Hastie C.J., Gray N.S, & Arthur J.S. (2021). Generation of a chemical genetic model for JAK3. Scientific Reports, 11, 10093.

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Isolating and Transducing Murine CD8+ T-cells

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T-cells were isolated from spleens of C57BL/6 mice and were enriched with a mouse CD8a+ T-cell isolation kit (#130-104-075, Miltenyi Biotec). Transduction of primary murine T-cells was conducted as previously described.^{14–16 (link)} In short, supernatants containing the virus of interest were used for transduction on two consecutive days (Supplementary Figure 1A-C). During virus generation, primary murine T-cells were activated with anti-CD28 (#16-0281-86, Thermo Fisher) and anti-CD3 antibodies (#16-0031-86, Thermo Fisher) in murine T-cell medium supplemented with IL-2 (#20-002, Peprotech) and β-mercaptoethanol (#21-9850-23, Gibco) for 24 hours. During the transduction process, T-cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 (#11-452D, Thermo Fisher). Transduced murine T-cells were expanded with murine T-cell medium and supplemented with IL-2 and β-mercaptoethanol, and maintained at a concentration of 1 × 10⁶ cells per mL every second day. Ex vivo-culture of T-cells was 4–7 days. Following retroviral transduction and expansion, ^EpCAM/GFPCAR T-cells were selected by GFP-positivity utilizing a MolFloll Cell Sorter (Beckman Coulter) after exclusion of dead cells.

Xu T., Karschnia P., Cadilha B.L., Dede S., Lorenz M., Seewaldt N., Nikolaishvili E., Müller K., Blobner J., Teske N., Herold J.J., Rejeski K., Langer S., Obeck H., Lorenzini T., Mulazzani M., Zhang W., Ishikawa-Ankerhold H., Buchholz V.R., Subklewe M., Thon N., Straube A., Tonn J.C., Kobold S, & von Baumgarten L. (2023). In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer. Oncoimmunology, 12(1), 2163781.

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Humanized Mouse Model for CTLA-4 Inhibitor Study

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Human PBMCs were purchased from LONZA (4W‐270C). PBMCs analyzed in this study were obtained from two healthy donors; PBMCs were stimulated overnight with 5 µg mL⁻¹ anti‐CD3 (ThermoFisher) and 5 µg mL⁻¹ anti‐CD28 (ThermoFisher) mAb. Recipient 12‐week‐old NSG mice were irradiated with 2.5 Gy 24 h before PBMC injection. After overnight stimulation, PBMCs were washed with PBS and 6 × 10⁶ cells were resuspended in 200 µL PBS. PBMCs (200 µL) were injected intravenously into NSG mice. 200 µm of dNP2‐ctCTLA‐4 was injected intraperitoneally on days 0–4. On day 5, splenocytes were isolated and analyzed using a FACS Canto II flow cytometer. Studies of human naive T cells and human PBMCs were exempted by the Institutional Review Board of Hanyang University.

Kim G., Kim W., Lim S., Lee H., Koo J., Nam K., Kim S., Park S, & Choi J. (2021). In Vivo Induction of Regulatory T Cells Via CTLA‐4 Signaling Peptide to Control Autoimmune Encephalomyelitis and Prevent Disease Relapse. Advanced Science, 8(14), 2004973.

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T Cell Differentiation Induction

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Naive CD4⁺ T cells from the spleen of naive C57BL/6 mice were purified by positive selection and magnetic separation (Miltenyi Biotec, Auburn, CA, USA). The cells were treated with anti-CD3 (2 μg/mL; Thermo Fisher Scientific) and anti-CD28 (2 μg/mL; Thermo Fisher Scientific) antibodies in the presence of IL-12 (40 ng/mL; PeproTech, Rocky Hill, NJ, USA) and anti-IL-4 (5 μg/mL; Thermo Fisher Scientific) to induce Th1 cell differentiation and TGF-β (10 ng/mL; PeproTech) to induce Treg cell differentiation. Th1 and Treg cell differentiation were assessed 4 days after induction.

Gu Y., Zhou H., Yu H., Yang W., Wang B., Qian F., Cheng Y., He S., Zhao X., Zhu L., Zhang Y., Jin M, & Lu E. (2021). miR-99a regulates CD4+ T cell differentiation and attenuates experimental autoimmune encephalomyelitis by mTOR-mediated glycolysis. Molecular Therapy. Nucleic Acids, 26, 1173-1185.

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Coculture of MSCs and PBMCs for T-cell Analysis

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MSCs (1 × 10⁴) were plated in 48‐well plates in DF12 (C11330500BT, Thermo Fisher Scientific) with 10% fetal calf serum (10099141C, Thermo Fisher Scientific) and 1% penicillin–streptomycin (SV30010, Cytiva) for overnight culture. PBMCs were isolated from human peripheral blood using the Ficoll–Paque method, labeled with eBioscience Cell Proliferation Dye eFluor 450 (65‐0842‐90, Thermo Fisher Scientific, USA), and plated at 1 × 10⁵ cells per well in the same plate treated with the presence of 1 µg mL⁻¹ anti‐CD3 (16‐0037‐81, Thermo Fisher Scientific, USA) and anti‐CD28 (16‐0289‐81, Thermo Fisher Scientific, USA). After four days of co‐culture, cells in suspension were collected by gently washing with PBS and then labeled with antibodies against CD4 (PE/Dazzle 594 anti‐human CD4, 562989, Biolegend) and CD8 (PerCP‐Cy5.5 Mouse Anti‐Human CD8, 560662, BD Biosciences) for flow cytometric analysis.

Chen H., Wen X., Liu S., Sun T., Song H., Wang F., Xu J., Zhang Y., Zhao Y., Yu J, & Sun L. (2022). Dissecting Heterogeneity Reveals a Unique BAMBIhighMFGE8high Subpopulation of Human UC‐MSCs. Advanced Science, 10(1), 2202510.

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Quantifying T Cell Proliferation Assay

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Splenocytes were isolated from Lck-Cre Atg7^f/f or Atg7^f/f mice and activated with anti-CD3e (1 μg/mL) and anti-CD28 (0.5 μg/mL) (both from ThermoFischer) for 72 h and incubated with 0.5 μCi/well ³H-thymidine (Perkin Elmer, The Netherlands) for the last 16 h. The amount of ³H-thymidine incorporation was measured using a liquid scintillation analyzer (Tri-Carb 2900R). Responses are expressed as the mean disintegrations per minute (dpm). The stimulation index (s.i.) was defined by dividing the dpm under activated conditions by the dpm under non-activated conditions per mouse.

Amersfoort J., Douna H., Schaftenaar F.H., Foks A.C., Kröner M.J., van Santbrink P.J., van Puijvelde G.H., Bot I, & Kuiper J. (2018). Defective Autophagy in T Cells Impairs the Development of Diet-Induced Hepatic Steatosis and Atherosclerosis. Frontiers in Immunology, 9, 2937.

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