Anti cd28

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

Anti-CD28 is a monoclonal antibody that binds to the CD28 receptor on the surface of T cells. CD28 is a co-stimulatory molecule that plays a crucial role in the activation and proliferation of T cells. Anti-CD28 can be used as a laboratory tool to study T cell biology and immune responses.

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Lab products found in correlation

Anti cd3, by Miltenyi Biotec (8 mentions) Interleukin 2 (il 2), by R&D Systems (2 mentions) Anti cd3 antibody, by Miltenyi Biotec (2 mentions) Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions) Anti cd3, by Thermo Fisher Scientific (2 mentions) Live dead kit, by Thermo Fisher Scientific (2 mentions) Easysep human cd4 t cell enrichment kit, by STEMCELL (2 mentions) Protamine, by Merck Group (1 mentions) Gt551 h3 medium, by Takara Bio (1 mentions) Mouse tcrβ specific antibody, by BD (1 mentions) Lipopolysaccharides (lps), by Miltenyi Biotec (1 mentions) Fixable viability dye, by Thermo Fisher Scientific (1 mentions) Monensin solution, by Thermo Fisher Scientific (1 mentions) Anti cd49d, by BD (1 mentions) X vivo 15 medium, by Lonza (1 mentions) Anti cd3 clone okt3, by Miltenyi Biotec (1 mentions) T cell activation expansion kit, by Miltenyi Biotec (1 mentions) Rpmi 1640 medium, by PAN Biotech (1 mentions) Facsaria 2 cell sorter, by BD (1 mentions) Anti cd4 fitc, by BioLegend (1 mentions)

Spelling variants of this product

Anti-CD3/CD28 beads (33 citations) Anti-CD28 antibody (9 citations) Anti-CD3/CD28 monoclonal antibodies (9 citations) Anti-CD3/CD28 microbeads (6 citations) Anti-CD3/anti-CD28 beads (5 citations) Functional anti-human CD28 mAb (4 citations) Anti-CD2/anti-CD3/anti-CD28 beads (3 citations) Anti‐CD2/CD3/CD28 microbeads (3 citations) Soluble anti-CD28 antibodies (2 citations) Anti-CD28 antibodies (15E8, (2 citations)

21 protocols using anti cd28

Library Generation of Engineered TCRs

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After bulk TCR sequencing, top rank alpha chains and beta chains were selected and randomly paired to obtain a library of 116 candidate TCR constructs. Detailed information on the library can be found in Table S3. The TCR library was cloned into lentiviral vectors for T‐cell transduction. Multiplicity of infection (MOI) was controlled at 0.2 to ensure single lentivirus integration into the genome of each transduced T cell.
Lentiviruses were generated using the pMD2.G and pSPAX2 envelope plasmids in HEK293T cells. Freshly isolated PBMCs were activated with anti‐CD3 and anti‐CD28 (Miltenyi Biotec) overnight, and proceeded to virus infection. Viral supernatant mixed with 400 U/ml IL‐2 (R&D System, USA) and 8 μg/ml protamine (Sigma Aldrich) was added onto peripheral blood lymphocytes (PBLs) and centrifuged at 800 g and 25°C for 1.5 h. Transduced T cells were maintained in GT551‐H3 medium (TaKaRa) supplied with 5% human serum (GemCell), 1% penicillin/streptomycin (HyClone), and 400 IU/ml IL‐2. The expression of TCR genes was routinely examined using a mouse TCRβ‐specific antibody (BD Biosciences) by FACS.

Jiang J., Xia M., Zhang L., Chen X., Zhao Y., Zeng C., Yang H., Liang P., Li G., Li N., Qi H., Wei T, & Ren L. (2022). Rapid generation of genetically engineered T cells for the treatment of virus‐related cancers. Cancer Science, 113(11), 3686-3697.

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Isolation and Activation of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were separated from venous blood samples obtained from 25 healthy genotyped controls by Ficoll-Hypaque density-gradient centrifugation. The separated PBMCs were placed in 96 well-plates (2 × 10⁶ cells/well), and cultured in RPMI 1640 medium. PBMCs were treated with anti-CD3 antibody (Miltenyi Biotec, Palo Alto, CA) and anti-CD28 (Miltenyi Biotec, Palo Alto, CA) antibody (5:1) to simulate antigen presentation for 3 days, or were cultured in LPS (Miltenyi Biotec, Palo Alto, CA) to simulate an inflammatory signal (100ng/mL, Sigma, Missouri, USA) for 1 day.

Jiang Y., Wang H., Yu H., Li L., Xu D., Hou S., Kijlstra A, & Yang P. (2016). Two Genetic Variations in the IRF8 region are associated with Behçet’s disease in Han Chinese. Scientific Reports, 6, 19651.

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CD107a Expression in T-cell Activation

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T-cells were incubated in 96-well plates (80,000 cells/well), together with an equal amount of THP-1 or KOx3 cells, in the presence or not of functional grade anti-CD3 (clone OKT3, Miltenyi Biotech, Köln, Germany) at a concentration of 7.5 µg/ml. Co-cultures were maintained in a final volume of 100 µl of X-Vivo-15 medium (Lonza, Basel, Switzerland) for 6 hours at 37 °C with 5% CO₂. CD107a staining was done during cell stimulation, by the addition of an APC-conjugated anti-CD107a antibody (BD Biosciences, San Jose, California) at the beginning of the co-culture, together with 1 µg/ml of anti-CD49d (BD Biosciences, San Jose, California), 1 µg/ml of anti-CD28 (Miltenyi Biotech, Köln, Germany), and 1× Monensin solution (eBioscience, San Diego, California). After the 6 hours incubation period, cells were stained with a fixable viability dye (eBioscience, San Diego, California) and PE-conjugated anti-CD8 (Miltenyi Biotech, Köln, Germany) and analyzed by flow cytometry.

Galetto R., Lebuhotel C., Poirot L., Gouble A., Toribio M.L., Smith J, & Scharenberg A. (2014). Pre-TCRα supports CD3-dependent reactivation and expansion of TCRα-deficient primary human T-cells. Molecular Therapy. Methods & Clinical Development, 1, 14021-.

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In Vitro Differentiation of Naive CD4+ T Cells into Th17 Lymphocytes

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The naive CD4+ fraction was isolated using CD4 M-pluriBeads^® anti-hu (pluriSelect Life Science, Leipzig, Germany) from the buffy coats obtained from healthy, anonymous donors (buffy coats were purchased from the Regional Center for Blood Donation and Blood Treatment, Lodz, Poland). To differentiate naive CD4+ cells into human Th17 lymphocytes, the protocol by Wilson et al. [77 (link)] was used: cells were cultured in RPMI 1640 medium (PAN-Biotech, Aidenbach, Germany) containing 1% human AB serum and were treated with the following cytokines from PeproTech (Rocky Hill, NJ, USA): 50 ng/mL human IL-1b, 30 ng/mL human IL-6, 10 ng/mL human IL-23, 10 ng/mL human TGF-β, and beads coated with anti-CD2, anti-CD3, and anti-CD28 (T cell activation/expansion kit from Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 days.

Pastwińska J., Karaś K., Sałkowska A., Karwaciak I., Chałaśkiewicz K., Wojtczak B.A., Bachorz R.A, & Ratajewski M. (2022). Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity. International Journal of Molecular Sciences, 23(3), 1906.

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Th17 Cell Differentiation from Naive CD4+ T Cells

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CD4⁺ T cells were purified from spleen and lymph nodes of C57BL/6 mice using anti-CD4 microbeads (Miltenyi Biotech) and then stained in PBS with 2% FCS for 15 min at room temperature with anti-CD4-FITC and anti-CD62L-PercP (both Biolegend, San Diego, CA, USA). The naive CD4⁺CD62L^high T cells were sorted using the BD FACSAria II cell sorter. After sorting, cells were activated for Th17 differentiation with anti-CD3 (2 μg/ml, plate-bound), anti-CD28 (2 μg/ml, soluble), rhTGF-β1 (2.5 ng/ml, Miltenyi Biotec), and rmIL-6 (20 ng/ml, Miltenyi Biotec). Cells were cultured for 72 h and analyzed by FACS.

Talbot J., Peres R.S., Pinto L.G., Oliveira R.D., Lima K.A., Donate P.B., Silva J.R., Ryffel B., Cunha T.M., Alves-Filho J.C., Liew F.Y., Louzada-Junior P, & de Queiroz Cunha F. (2018). Smoking-induced aggravation of experimental arthritis is dependent of aryl hydrocarbon receptor activation in Th17 cells. Arthritis Research & Therapy, 20, 119.

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T-cell and B-cell Activation Assay

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For T-cell activation, 1.75 × 10⁵ T cells (stained with 0.2 µM CellTrace^TM violet (CTV)) per well were pretreated with zotatifin, CR-31-B (−), CR-31-B (+) or vehicle (DMSO) for 30 min in RPMI 1640 medium supplemented with 2 mM of glutamine, 50 units/mL of IL2, 1% penicillin/streptomycin and 10% FCS. Afterward, they were seeded in anti-CD3 (BioLegend, Fell, Germany) coated 96-well plates and stimulated with 2 µg/mL of anti-CD28 (Miltenyi Biotec, Bergisch Gladbach, Germany) for 5 days. CTV staining was performed according to the protocol of the supplier.
B cells were activated as described by Khoenkhoen et al. [38 (link)]. First, 1.25 × 10⁵ of B cells (stained with 0.25 µM of CTV) per well were seeded in a 48-well plate and pretreated with zotatifin, CR-31-B (−), CR-31-B (+) or vehicle (DMSO) for 30 min. Then, the B cells were stimulated with 5 μg/mL of unconjugated goat anti-human IgM F(ab’)2 fragments, 2.5 μg/mL of CpG, 1 μg/mL of sCD40L and 50 ng/mL of recombinant human IL21 for 5 days.

Schiffmann S., Henke M., Seifert M., Ulshöfer T., Roser L.A., Magari F., Wendel H.G., Grünweller A, & Parnham M.J. (2023). Comparing the Effects of Rocaglates on Energy Metabolism and Immune Modulation on Cells of the Human Immune System. International Journal of Molecular Sciences, 24(6), 5872.

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Regulatory T Cell Suppression Assay

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CD4⁺CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec, Auburn, CA) was used to isolate cells. The median purity of isolation procedures was >90%. CD4+CD25− T cells were stained with 1.5μM CFSE (Molecular Probes/Invitrogen, Carlsbad, CA) for 10 min at room temperature. After quenching by FBS, cells were washed with PBS. A total of 5×10⁴ of CD4+CD25+ cells were cultured in triplicate with 5×10⁴ CFSE-labelled autologous CD4⁺CD25⁻ T cells stimulated with anti-CD3 (1 μg/mL) (ATCC, Rockville, MD) and anti-CD28 Abs (1 μg/mL) (Miltenyi Biotec, Auburn, CA) in AIM V medium supplemented with IL-2 at 150 IU/mL. As negative controls, CFSE-labelled CD4+CD25-cells were stimulated with anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) in the absence of fresh CD4+CD25+ cells. CFSE expression was measured by flow cytometry on day five after stimulation. CFSE data were analyzed using the ModFit software. The percentages of suppression were calculated based on the proliferation index (PI) of responder cells alone compared with the PI of cultures containing responders and Treg. The program determines the percent of cells within each peak, and the sum of all peaks in the control culture is taken as 100% of proliferation and 0% of suppression.

Geskin L.J., Akilov O.E., Kwon S., Schowalter M., Watkins S., Whiteside T.L., Butterfield L.H, & Falo LD J.r. (2017). Therapeutic reduction of cell-mediated immunosuppression in mycosis fungoides and Sézary syndrome. Cancer immunology, immunotherapy : CII, 67(3), 423-434.

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Activation and Proliferation of Mouse CD4+ and CD8+ T Cells

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CD4⁺ T cells were positively selected from mouse spleens using magnetic beads (Miltenyi, catalog #130–049-201) and stimulated with 10 μg/mL plate-bound anti-CD3 (eBioscience, catalog #16–0031-85) and 2 μg/mL anti-CD28 (eBioscience, catalog #16–0281-85) in the presence of 250 nM APG-115 or DMSO for 1 or 2 days. After treatment, cells were harvested and evaluated by flow cytometry for the expression of CD25 (BD, catalog #557192), CD62L (BD, catalog #553151), and Foxp3 (Thermo Fisher, catalog #12–5773-82). T cell activation were defined as CD25^highCD62L^low and enlarged cell size. CD25⁺Foxp3⁺T cells represented Treg population.
For T cell proliferation, CD4⁺ T and CD8⁺ T cells were positively selected from mouse spleens using magnetic beads (Miltenyi, catalog #130–049-201 and #130–096-495) and then stimulated with a series of concentrations of plate-bound anti-CD3 and 2 μg/mL anti-CD28 in the presence of 250 nM APG-115 or DMSO. After 72 h, relative cell numbers were determined using CellTiter-Glo luminescent cell viability assay (Promega, catalog #G7571) and normalized to unstimulated cultures treated with DMSO control.

Fang D.D., Tang Q., Kong Y., Wang Q., Gu J., Fang X., Zou P., Rong T., Wang J., Yang D, & Zhai Y. (2019). MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment. Journal for Immunotherapy of Cancer, 7, 327.

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Retroviral transduction of primary T cells

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The 293GP packaging line was plated overnight onto poly-d-lysine-coated 60-mm² plates at 1.6 × 10⁶ cells per plate in complete DMEM. 293GP cells were transfected with 6 μg of a candidate TCR pMSGV1-plasmid along with 3 μg of the RD114 envelope using the Lipofectamine 3000 (Invitrogen) reagent set. Both the pMSGV1 and RD114 plasmids were obtained through an MTA from S. A. Rosenberg. Viral supernatant was collected after 48 hours and loaded onto retronectin-coated (10 ug ml⁻¹, Takara Bio) non-TC 24-well plates and centrifuged at 2,000g for 2 hours at 32 °C. CD8⁺ and CD4⁺ T cells enriched from HD polyclonal PBMCs were stimulated with plate-bound OKT3 (5 μg ml⁻¹, Miltenyi Biotec), anti-CD28 (2 μg ml⁻¹, Miltenyi Biotec) and rhIL-2 (300 IU ml⁻¹) for 48 hours before transduction. Stimulated cells were plated at 1–5 × 10⁵ cells per well and spinoculated at 1,500 r.p.m. for 15 minutes. Cells were assessed for transduction efficiency after 3–4 days by measuring surface expression of mTCR by FACS.

Chandran S.S., Ma J., Klatt M.G., Dündar F., Bandlamudi C., Razavi P., Wen H.Y., Weigelt B., Zumbo P., Fu S.N., Banks L.B., Yi F., Vercher E., Etxeberria I., Bestman W.D., Da Cruz Paula A., Aricescu I.S., Drilon A., Betel D., Scheinberg D.A., Baker B.M, & Klebanoff C.A. (2022). Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA. Nature Medicine, 28(5), 946-957.

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CD15+ Cell Depletion and T Cell Proliferation

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Peripheral blood cells were labeled with an anti‐CD15‐APC antibody and anti‐APC magnetic beads (Miltenyi Biotec K.K., Tokyo, Japan). The CD15⁺ cells were depleted from PBC using MiniMACS columns (Miltenyi Biotec). The control PBC were labeled with anti‐mouse IgG1‐APC antibody and anti‐APC magnet beads. In other experiments, the sorted CD15⁺ cells were added to the PBC at up to 10 times higher concentration than in original PBC. PBC with and without CD15⁺ cells were labeled with carboxy‐fluorescein diacetate succinimidyl ester (CFSE) (Cayman Chemichal, Ann Arbor, MI, USA) or CellTrace Violet (CTV) (Invitrogen, Carlsbad, CA, USA), and were stimulated with anti‐CD3 (5 μg/mL) (eBioscience, San Diego, CA, USA) and anti‐CD28 (5 μg/mL) (Miltenyi Biotec) antibodies in RPMI 1640 medium. In some experiments H₂O₂ scavenger catalase (100 U/mL, catalase from bovine liver; Sigma‐Aldrich) was added into the T cell culture. After a 48‐h or 72‐h incubation, the cells were collected and stained with anti‐CD3‐PE, CD4‐APC and CD8‐APC antibodies for flow cytometric analysis. The proliferation of CD4⁺ and CD8⁺ T cells was assessed by CFSE or CTV fluorescence intensity.

Horinaka A., Sakurai D., Ihara F., Makita Y., Kunii N., Motohashi S., Nakayama T, & Okamoto Y. (2016). Invariant NKT cells are resistant to circulating CD15+ myeloid‐derived suppressor cells in patients with head and neck cancer. Cancer Science, 107(3), 207-216.

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