Anti cd28 antibody

Manufactured by BD

Sourced in United States, Germany

The Anti-CD28 antibody is a laboratory reagent used in immunology research. It binds to the CD28 receptor on the surface of T cells, a key co-stimulatory molecule involved in T cell activation.

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Spelling variants of this product

Anti-CD28 (379 citations) Soluble anti-CD28 antibody (15 citations) CD28 antibodies (11 citations) Anti-CD3/anti-CD28 antibodies (9 citations) Anti-CD28 monoclonal antibody (5 citations) Anti-mouse CD28 antibody (3 citations) Anti-CD28 antibody (clone 37.51; (3 citations) Mouse anti-human CD28 monoclonal antibody (2 citations) Mouse anti-human CD28 antibody (2 citations) Anti-CD28 antibody (37.51) (2 citations)

65 protocols using anti cd28 antibody

Modulation of IFN-γ and anti-dsDNA IgG

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A total of 1–2 × 10⁶ cells isolated from lymph nodes were plated in complete culture medium (Dulbecco’s Modified Eagle Medium (DMEM), 10% FBS, 1% antibiotic–antimycotic (ABAM), and 0.1% beta-mercaptonol) with or without 3 μg/ml anti-CD3 and 3 μg/ml anti-CD28 antibodies (BD Biosciences, San Diego, CA), 20 μM SOCS1-KIR, 20 μM SOCS1-KIR Dimer, and 20 μM pJAK2 mimetic peptides. IFN-γ was measured with an ELISA kit (BD Pharmingen) as previously described^{41 (link)} on supernatants harvested after a 5 days incubation.
Serum was collected from animals weekly to assess the production of anti-dsDNA IgG. Anti-dsDNA IgG was measured in sera diluted 1:1000, 1:3000, or 1:9000 in plates coated with 50 μg/mL dsDNA. Bound IgG was detected using alkaline phosphatase-conjugated anti-mouse IgG diluted 1:1000. The absorbance at 450 nm was measured.

Sharma J., Collins T.D., Roach T., Mishra S., Lam B.K., Mohamed Z.S., Veal A.E., Polk T.B., Jones A., Cornaby C., Haider M.I., Zeumer-Spataro L., Johnson H.M., Morel L.M, & Larkin J I.I.I. (2021). Suppressor of cytokine signaling-1 mimetic peptides attenuate lymphocyte activation in the MRL/lpr mouse autoimmune model. Scientific Reports, 11, 6354.

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Immune Response Assessment in Murine Splenocytes and Macrophages

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Single-cell suspensions from spleens of mice in animal experiment 1 were prepared passing cells through a 100 μM strainer. The splenocytes were plated at a density of 1 × 10⁶/ml and stimulated with 2 μg/ml anti-CD3 and 1 μg/ml anti-CD28 antibodies (BD PharMingen, San Diego, CA, USA). The peritoneal macrophages from mice in animal experiment 1 were cultured in the presence of 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO) in DMEM at a density of 1 × 10⁶/ml. The cells were cultured at 37°C for 24 hours before supernatant collection. Supernatants from splenocyte cultures were collected and analyzed by ELISA for INF-γ. The supernatants from peritoneal macrophage cultures were analyzed for TNF-α. All ELISA kits were purchased from eBioscience Inc. (San Diego, CA, USA).

Wang S., Ye Q., Wang K., Zeng X., Huang S., Yu H., Ge Q., Qi D, & Qiao S. (2019). Enhancement of Macrophage Function by the Antimicrobial Peptide Sublancin Protects Mice from Methicillin-Resistant Staphylococcus aureus. Journal of Immunology Research, 2019, 3979352.

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Isolation and Activation of Murine T Cells

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Spleens were isolated from control BALB/c mice under aseptic conditions and prepared for cellular suspensions through a 70 μm cell strainer (BD Biosciences, San Jose, CA, USA). Single-cell suspensions were resuspended in an erythrocyte-lysing buffer (BD Biosciences) and washed. T cells were enriched by a Pan T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s specifications. The purity (CD3ε⁺) was confirmed at > 90% by flow cytometry. Normal T cells were stimulated with 3 μg/ml anti-CD3ε plus 500 ng/ml anti-CD28 antibodies (BD Biosciences) and cultured in RPMI 1640 (Life Technologies) without l-arginine for 24 h. Media was supplemented with 4% fetal bovine serum (Gibco, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin.

Cao S., Gong W., Zhang X., Xu M., Wang Y., Xu Y., Cao J., Shen Y, & Chen J. (2020). Arginase promotes immune evasion of Echinococcus granulosus in mice. Parasites & Vectors, 13, 49.

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CD8+ T Cell Activation and Cancer Cell Interaction

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Splenocytes derived from wild-type (WT) and Rab37 knockout (KO) mice were obtained by mashing the spleen and removing erythrocytes using red blood cells lysis buffer. CD8⁺ T cells were isolated using mouse CD8 T lymphocyte enrichment set (BD Bioscience, East Rutherford, NJ, United States). Purified CD8⁺ T cells were activated by either 100 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, Louis City, MO, United States) plus 100 ng/ml of Ionomycin (Io, Sigma-Aldrich) or 2 μg/ml anti-CD3 together with 2 μg/ml anti-CD28 antibodies (BD Bioscience) for 24 or 48 h. T cells with or without treatment with conditioned medium (CM) derived from cancer cells were collected and analyzed by immunofluorescence and flow cytometry.

Kuo W.T., Kuo I.Y., Hsieh H.C., Wu S.T., Su W.C, & Wang Y.C. (2024). Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer. Journal of Biomedical Science, 31, 20.

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Splenic γδ T Cell Activation

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Splenic γδ T cells were MACS isolated using “TCRg/d T-cell isolation kit, mouse” (Miltenyi Biotec). Cells numbering 300,000 were stimulated by plate-bound αnti-CD3 (10 μg/mL, BD Bioscience) and anti-CD28 antibodies (2 μg/mL, BD Bioscience) for 24 h at 37 °C. After stimulation, cells were stained for surface markers and analyzed by flow cytometry.

Seeland I., Xiong Y., Orlik C., Deibel D., Prokosch S., Küblbeck G., Jahraus B., De Stefano D., Moos S., Kurschus F.C., Arnold B, & Samstag Y. (2018). The actin remodeling protein cofilin is crucial for thymic αβ but not γδ T-cell development. PLoS Biology, 16(7), e2005380.

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Modulation of IFN-γ production by pDCs

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Freshly prepared PBMCs with or without pDC depletion were cultured for 5 days in a 24-well plate stimulated with anti-CD3 (5 μg/ml) (BD-PharMingen) and anti-CD28 antibodies (3 μg/ml) (BD-PharMingen) in the presence or absence of 10 ug/ml CpG (ODN2336) (Coley Pharmaceutical, Wellesley, MA). At day 5, the above culture was stimulated with T cell activation cocktails (PMA+ionomycin+GolgiPlug) (BD-PharMingen) for 4 h, the cells were stained with anti-CD4-PE or –APC, then underwent intracellular staining for IFN-γ following instructions from the manufacturer (BD-PharMingen). IFN-γ-producing CD4⁺ T cells were analyzed by flow cytometry by gating CD4⁺ T cells. In some experiments, CD4⁺CD45RA⁺ T cells were purified through negative selection using StemCell Sep kit (StemCell Technology, Vancouver, Canada). Different numbers of pDCs (5000, 2500, 1250, 0) purified by BDCA-2 microbeads were incubated with autologous CD4⁺CD45RA⁺ T cells stimulated with CD3 and CD28 antibodies in the presence or absence of CpG as described above. IFN-γ in the supernatants was measured using Luminex assay kit (Millipore, Billerica, MA) and IFN-γ-producing cells were examined by intracellular cytokine staining.

Xia C.Q., Peng R., Chernatynskaya A.V., Yuan L., Carter C., Valentine J., Sobel E., Atkinson M.A, & Clare-Salzler M.J. (2014). Increased IFN-α-producing Plasmacytoid Dendritic Cells (pDCs) in Human Th1-mediated Type 1 Diabetes: pDCs Augment Th1 Responses through IFN-α Production. Journal of immunology (Baltimore, Md. : 1950), 193(3), 1024-1034.

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Isolation and Expansion of Murine T Cells

Cited 1 time

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We generated pooled (nonsorted) single‐cell suspensions from phosphate buffered saline (PBS)–perfused spleen or CP tissue excised from donor 16‐ to 18‐week‐old female MRL/lpr mice (7–10 mice), as previously described (17 (link)). Cells were initially stimulated and expanded on plates precoated with 0.5 μg/ml of anti‐CD3 antibodies (BD Pharmingen) and with 0.5 μg/ml of anti‐CD28 antibodies (BD Pharmingen) added to RPMI 1640 medium supplemented with 10% fetal bovine serum (17 (link)). We then added 10 units of murine interleukin‐2 (IL‐2) (R&D Systems) every other day to further stimulate T cell proliferation for 7 days, after which cells were prepared for phenotyping and/or injection.

Moore E., Huang M.W., Reynolds C.A., Macian F, & Putterman C. (2022). Choroid Plexus–Infiltrating T Cells as Drivers of Murine Neuropsychiatric Lupus. Arthritis & Rheumatology (Hoboken, N.j.), 74(11), 1796-1807.

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T Cell Proliferation Assay with MSCs

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Lymphocytes isolated from peripheral lymph nodes of 8 to 10 weeks-old C57BL/6 mice were labeled with 5-μM CFSE (Beyotime) for 10 min, at 37°C, seeded at 2×10⁵ cells/100 μl/well in 1640 complete medium (RPMI-1640 + 10% FBS). MSC were seeded in a 6-well plate, cultured in the MSC medium for 7 days, dissociated with Trypsin-EDTA (StemCell Technologies), and added at the ratio of 1:40 or 1:80 (for cell number of MSC versus lymphocytes) per well into a 96-well plate containing lymphocytes at 100 μl/well. The lymphocytes in the mixture were stimulated with or without pre-coated 10-μg/ml anti-CD3 antibodies (BD Bioscience) and 2-μg/ml anti-CD28 antibodies (BD Bioscience). Suspended lymphocytes were collected 5 days after the stimulation and then subjected to flow cytometry analysis.

Yan L., Jiang B., Li E., Wang X., Ling Q., Zheng D., Park J.W., Chen X., Cheung E., Du X., Li Y., Cheng G., He E, & Xu R.H. (2018). Scalable Generation of Mesenchymal Stem Cells from Human Embryonic Stem Cells in 3D. International Journal of Biological Sciences, 14(10), 1196-1210.

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Murine Lymphocyte Proliferation Assay

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Lymphocytes derived from the lymph nodes of 10 week-old Kunming mice were first labeled with 5 µM CFDA SE Cell Proliferation Assay and Tracking Kit (Beyotime, Jiangsu, China) for 10 min in a 37 °C water bath and then cultured in 1640 complete medium (RPMI-1640 plus 10% FBS) in a 96-well plate at a density of 2 ×10⁵ cells/100 µL/well. Rhesus MSCs were expanded in a 6-well plate and cultured for five days. Then, the MSCs were dissociated with 0.05% Trypsin-EDTA and added to the 96-well plate cultured with lymphocytes. The ratio of MSCs to lymphocytes was 1:40 or 1:80 per well. The lymphocytes in the mixture were stimulated with or without pre-coated 10-µg/ml anti-CD3 antibodies (BD Biosciences, San Jose, CA, USA) and 2 µg/mL anti-CD28 antibodies (BD Biosciences, San Jose, CA, USA) or on beads. Lymphocytes were collected 5 days later after stimulation and then subjected to flow cytometry analysis. The data were analyzed with Flowjo (TreeStar, Ashland, OR, USA).

Fu X., Jiang B., Zheng B., Yan Y., Wang J., Duan Y., Li S., Yan L., Wang H., Chen B., Sang X., Ji W., Xu R.H, & Si W. (2018). Heterogenic transplantation of bone marrow-derived rhesus macaque mesenchymal stem cells ameliorates liver fibrosis induced by carbon tetrachloride in mouse. PeerJ, 6, e4336.

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B Cell and T Cell Coculture Assay

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B cells and CD4⁺ T cells were isolated with anti-mouse CD19⁺ microbeads and anti-mouse CD4⁺ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For mitogen stimulation, B cells were cultured with LPS (10 μg/ml, Sigma-Aldrich, St. Louis, MO, USA), anti-IgM F(ab′)₂ (30 μg/ml, Jackson Immunoresearch, West Grove, Pennsylvania, USA) or anti-CD40 antibodies (10 μg/ml, BD Bioscience, San Jose, CA, USA), and CD4⁺ T cells were cultured with anti-CD3 (4 μg/ml) and anti-CD28 antibodies (4 μg/ml). In the coculture assay, CFSE-labeled B cells and CD4⁺ T cells were cocultured on microtiter wells pre-coated with anti-CD3/CD28 antibodies. After 3 d of co-culture, the cells were stained with CD19-PE (eBioscience, San Diego, CA, USA) and CD138-APC (BD Bioscience, San Jose, CA, USA) to determine the percentage of plasma cells in CFSE⁺ B cells. After 7 d of coculture, the supernatant was collected to determine IgG production by ELISA (Bethyl Laboratories, Montgomery, TX, USA).

Liu Y.H., Tsai Y.S., Lin S.C., Liao N.S., Jan M.S., Liang C.T., Hsu S.W., Chen W.C., Sung J.M., Maeda N, & Tsai P.J. (2016). Quantitative PPARγ expression affects the balance between tolerance and immunity. Scientific Reports, 6, 26646.

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