Anti human cd28 cd28.2

Manufactured by Thermo Fisher Scientific

The Anti-human CD28 (CD28.2) is a monoclonal antibody that binds to the CD28 receptor found on the surface of T cells. CD28 is a costimulatory molecule that plays a crucial role in T cell activation and proliferation. This antibody can be used in various applications, such as flow cytometry, immunohistochemistry, and cell stimulation assays, to study the function and dynamics of T cells.

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Anti-CD28 (306 citations) Anti-human CD28 (6 citations) CD28 (CD28.2; (4 citations) Mouse anti-human CD28 (Clone 28.2; (2 citations) Purified anti-human CD28 (CD28.2) (2 citations)

3 protocols using anti human cd28 cd28.2

T-cell Activation Assay Protocol

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Cells were cultured in complete Dulbecco’s Modified Eagle’s medium (#10-013-CV, Corning) supplemented with 9% pooled GemCell U.S. Origin Human Serum AB (#100-512, GeminiBio), 2 mM L-glutamine (#25030081, Gibco), and 1% penicillin-streptomycin (#15140122, Gibco).
Functional grade purified anti-human CD3 (OKT3) and functional grade purified anti-human CD28 (CD28.2; eBioscience) were added in suspension at a concentration of 4 μg/mL together with 200 ng/mL recombinant human IL-2 (PeproTech).

Eskandari S.K., Allos H., Al Dulaijan B.S., Melhem G., Sulkaj I., Alhaddad J.B., Saad A.J., Deban C., Chu P., Choi J.Y., Kollar B., Pomahac B., Riella L.V., Berger S.P., Sanders J.S., Lieberman J., Li L, & Azzi J.R. (2022). mTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis. Frontiers in Immunology, 13, 899975.

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Immunoblotting and Immunofluorescence Analysis

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For immunoblotting, we used: anti-phospho Erk1/2 (D13.14.4E), p38 (D3F9), and Jnk (81E11), and anti-total Erk1/2 (137F5), p38 (#9212), Jnk (#9252) Abs; anti-phospho Zap70 (Tyr319) and PLC-γ1 (Tyr783) Abs (Cell Signaling Technology), anti-phospho Lat (Tyr191) and anti-p-Tyr (4G-10) Abs (Millipore); Anti-CD3ε (M-20; sc-1127), anti-Lck (2102; sc-13) and anti-TRPV1 (P-19; sc-12498) Abs (Santa Cruz Biotechnology); anti-TRPV1 (#ACC-030, Alomone), anti-NFAT1 mAb (ab2722, Abcam), and anti-β-actin (AC-74, Sigma). For immunofluorescence studies, anti-TRPV1 (sc-12498) and anti-Lck (sc-13) (Santa Cruz Biotechnology), anti-CD4 (GK1.5, eBioscience), anti-CD45 and anti-Claudin-3 Abs (BD Biosciences). For T cell stimulation, anti-mouse CD3ε (145-2c11) and anti-mouse CD28 (PV-1) (BioXcell), and anti-human CD3ε (UCHT1) and anti-human CD28 (CD28.2) (eBioscience) mAbs were used; PMA (Phorbol 12-myristate 13-acetate), ionomycin, thapsigargin and phytohemagglutinin were from Sigma; The Src-family kinase inhibitor PP2 was from Cayman Chemical, the TRPV1 antagonists were from Tocris (BCTC, I-RTX) and Enzo Life Sciences (SB366791), the Annexin-V/7-AAD apoptosis detection kit was from BD Biosciences and CFSE, Indo-1 AM, and Fura-2 AM were from Invitrogen/Molecular Probes.

Bertin S., Aoki-Nonaka Y., de Jong P.R., Nohara L.L., Xu H., Stanwood S.R., Srikanth S., Lee J., To K., Abramson L., Yu T., Han T., Touma R., Li X., González-Navajas J.M., Herdman S., Corr M., Fu G., Dong H., Gwack Y., Franco A., Jefferies W.A, & Raz E. (2014). The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells. Nature immunology, 15(11), 1055-1063.

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Antibody Characterization and Validation

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Antibodies to SENP1 (C-12), Lck (3A5), RanGAP1 (C-5), c-Myc (9E10), β-actin (C4), and GAPDH (FL-335) were from Santa Cruz Biotechnology. Antibodies to SENP1 (ab108981), SUMO1 (ab32058), and SUMO2/3 (ab3742) were from Abcam. Antibodies to HA (C29F4) and glutathione S-transferase (GST) were from Cell Signaling Technology. M2 antibody to Flag (F1804) was from Sigma-Aldrich, and 4G10 (05-1050) was from Millipore. Anti-human CD3 (OKT3) and anti-human CD28 (CD28.2) were from eBioscience. Goat anti-mouse IgG (31160) was from Thermo Fisher Scientific. Horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch.

Li Y.Y., Cen H., Gong B.N., Mai S., Wang Q.L., Mou S, & Li Y. (2022). TCR-Induced Tyrosine Phosphorylation at Tyr270 of SUMO Protease SENP1 by Lck Modulates SENP1 Enzyme Activity and Specificity. Frontiers in Cell and Developmental Biology, 9, 789348.

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