Soluble anti mouse cd28

Manufactured by BD

Sourced in United States

Soluble anti-mouse CD28 is a laboratory reagent that binds to the CD28 receptor on the surface of mouse T cells. CD28 is a costimulatory molecule that plays a crucial role in T cell activation and proliferation. This reagent can be used in cell culture and immunological assays involving mouse T cells.

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Anti-CD28 (379 citations) Soluble anti-CD28 (89 citations) Anti-mouse CD28 (28 citations) Mouse anti (2 citations)

4 protocols using soluble anti mouse cd28

Isolation and Activation of CD8+ T Cells

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Spleens isolated from P14, Pmel, or OT-1 TCR transgenic mice were dissociated in complete RPMI media [RPMI 1640 (Gibco) + 10% fetal bovine serum (FBS; Gibco) + 1% penicillin-streptomycin (Gibco)], and red blood cells were lysed using RBC lysis buffer (BioLegend). CD8⁺ T cells were isolated using a CD8a⁺ T cell isolation kit (Miltenyi Biotec). For T cell activation, isolated CD8 T cells were cultured in T cell media [complete RPMI media supplemented with 1× nonessential amino acids (Gibco) + 1 × 10⁻³ M sodium pyruvate (Gibco) + 0.05 × 10⁻³ M 2-mercaptoethanol (Sigma-Aldrich)] supplemented with soluble anti-mouse CD28 (5 μg/ml; BD Pharmingen) and rhIL-2 (30 U/ml; Roche) at 1 × 10⁶ cells/ml in wells coated with anti-mouse CD3e (3 μg/ml; BD Pharmingen).

Su F.Y., Zhao Q.H., Dahotre S.N., Gamboa L., Bawage S.S., Silva Trenkle A.D., Zamat A., Phuengkham H., Ahmed R., Santangelo P.J, & Kwong G.A. (2022). In vivo mRNA delivery to virus-specific T cells by light-induced ligand exchange of MHC class I antigen-presenting nanoparticles. Science Advances, 8(8), eabm7950.

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Isolation and Activation of OT-I T Cells

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OT-I T cells were isolated from the spleens of OT-I T cell receptor transgenic mice using the CD8α⁺ T cell isolation mouse kit (Miltenyi), as per manufacturer’s instructions. Purified OT-I T cells were then stimulated in 24-well plates with plate-bound anti-mouse CD3ε (2 μg ml⁻¹, BD Biosciences), soluble anti-mouse CD28 (2 μg ml⁻¹, BD Biosciences) and recombinant human IL-2 (100 U ml⁻¹, DFCI supply centre). After 48 h, activated OT-I T cells were transferred into fresh media containing recombinant human IL-2 and allowed to expand for 5–7 days. For restimulation, 1 × 10⁵ OVA-expressing Ptpn2-null or control sgRNA-transfected B16 tumour cells were first plated in 24-well plates and stimulated with recombinant mouse IFNγ overnight to induce ovalbumin surface expression. 1 × 10⁶ pre-activated OT-I T cells were then added to the wells on the next day and co-cultured with the B16 tumour cells for 2–3 h. Subsequently, 1× brefeldin (eBiosciences) was added to the cultures for 4 h to inhibit intracellular protein transport. Afterward, OT-I T cells were collected from each well, stained for surface markers and then fixed with Foxp3/Transcription Factor Staining Buffer Set (eBiosciences) as per the manufacturer’s instructions. Intracellular cytokine staining was then performed before analysis on an LSR Fortessa.

Manguso R.T., Pope H.W., Zimmer M.D., Brown F.D., Yates K.B., Miller B.C., Collins N.B., Bi K., LaFleur M.W., Juneja V.R., Weiss S.A., Lo J., Fisher D.E., Miao D., Van Allen E., Root D.E., Sharpe A.H., Doench J.G, & Haining W.N. (2017). In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature, 547(7664), 413-418.

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Naive CD4+ T Cell Isolation and Culture

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Treat C57BL/6 mouse spleen tissue, crush on a cell filter to obtain a single cell suspension, and lyse erythrocytes. Naïve CD4+ T cells were sorted according to the MagniSort™Mouse CD4 Naïve T cell Enrichment Kit supplier time instructions (Invitrogen, Carlsbad, CA, USA).
For all T cell cultures, RPMI 1640 (Cytiva, Marlborough, MA, USA) medium was supplemented with 10% FBS (GIBCO, Grand Island, NY, USA), 10 mM HEPES buffer (GIBCO, Grand Island, NY, USA), 55 mM 2-ME (GIBCO, Grand Island, NY, USA), antibiotic–antifungal (GIBCO, Grand Island, NY, USA) and 2 mM L -glutamine (GIBCO, Grand Island, NY, USA), 1 mM sodium pyruvate (GIBCO, Grand Island, NY, USA) and MEM non-essential amino acids (GIBCO, Grand Island, NY, USA). About 100,000 cells were added to 96-well plates coated with anti-mouse CD3 (2.5 mg/mL, BD Pharmingen, San Diego, CA, USA) and soluble anti-mouse CD28 (2 mg/mL, BD Pharmingen, San Diego, CA, USA). T cells were cultured with CCL2 or CCL8 (MedChem Express, Monmouth Junction, NJ, USA).

Jin R., Zhou M., Lin F., Xu W, & Xu A. (2023). Pathogenic Th2 Cytokine Profile Skewing by IFN-γ-Responding Vitiligo Fibroblasts via CCL2/CCL8. Cells, 12(2), 217.

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Naïve CD4+ T Cell Differentiation into Th17 Cells

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Naïve CD4 + T cells were purified from mouse spleen samples using magnetic beads (Naive CD4 + T Cell Isolation Kit, mouse; Miltenyi Biotec, Gladbach, Germany). Naïve CD4 + T cells were primed with plate-bound anti-CD3 (1 µg/ml) (BD Biosciences PharMingen, San Diego, CA, USA) and soluble anti-mouse CD28 (2 µg/ml) (BD Biosciences PharMingen) in round bottomed 96well plates with RPMI1640 medium containing a 10% foetal bovine serum and 1% penicillin/streptomycin solution (Hyclone, Logan, UT, USA). For Th17 differentiation, the cells were cultured in the presence of soluble anti-CD28 (1 μg/ml), rmIL-2 (20 ng/ ml; Peprotech, London, UK), rmTGF-β (5 ng/ml; R&D Systems, Minneapolis, MN, USA), rmIL-6 (25 ng/ml; Peprotech), and anti-IL-4 and anti-IFN-γ neutralizing antibodies (10 μg/ml, Biolegend) for 5 days. The cells were then washed with PBS, transferred to a new plate to incubate for 2 days, and were then re-stimulated for 40 h with Dynabeads Mouse T-Activator CD3/CD28 (Invitrogen, Carlsbad, CA, USA). For the chemical inhibition, the following chemicals were then added to the sample medium: Ceranib-1 (30 μM; ( 21

Shin S.H., Cho K.A., Hahn S., Lee Y., Kim Y.H., Woo S.Y., Ryu K.H., Park W.J, & Park J.W. (2019). Inhibiting Sphingosine Kinase 2 Derived-sphingosine-1-phosphate Ameliorates Psoriasis-like Skin Disease via Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes in Mice. Acta dermato-venereologica, 99(6).

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