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Anti cd28 and anti cd49d co stimulatory antibodies

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Anti-CD28 and anti-CD49d co-stimulatory antibodies are laboratory reagents used to activate and stimulate T cells. These antibodies bind to specific cell surface receptors, CD28 and CD49d, which are involved in the co-stimulation of T cell activation and proliferation. The core function of these antibodies is to provide co-stimulatory signals to enhance T cell responses in various in vitro and ex vivo applications.

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Lab products found in correlation

Foetal calf serum, by Merck Group (1 mentions) Dimethyl sulphoxide, by Merck Group (1 mentions) Purified protein derivative, by Statens Serum Institut (1 mentions) Staphylococcus enterotoxin b, by Merck Group (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Iscove s modified dulbecco s medium, by Lonza (1 mentions) Anti ifn γ apc, by BD (1 mentions) Anti tnf α pe, by BD (1 mentions) Anti cd4 v500, by BD (1 mentions) 7 aad, by BD (1 mentions) Software, by FlowJo (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Brefeldin a, by BD (1 mentions) Cytofix cytoperm buffer, by BD (1 mentions) Anti cd3 fitc, by BD (1 mentions) Anti cd8 pe cy7, by BD (1 mentions) Brefeldin a, by Merck Group (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Facs lysing solution, by BD (1 mentions)

3 protocols using anti cd28 and anti cd49d co stimulatory antibodies

1

PBMC Stimulation and Cytokine Analysis

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PBMC were isolated and stimulated as previously described [24 (link)] or in some experiments, venous blood was diluted 1:1 with warm Iscove’s Modified Dulbecco’s Medium (Lonza, Belgium) and stimulated directly. Where frozen PBMC were thawed and used, these were previously cryopreserved at -80°C in freezing medium (RPMI 1640 (Invitrogen) + 20% foetal calf serum (Sigma) + 10% dimethylsulphoxide (Sigma)). PBMC (fresh or thawed) or diluted whole blood was stimulated for between 18 hours and five days in different experiments with heparin-binding haemaggluttinin (HBHA) (10μg/ml; purified as previously described [25 (link)]), recombinant ESAT-6 protein (Lionex, Braunschweig, Germany; 10μg/ml), purified protein derivative (PPD; Statens Serum Institute, Copenhagen, Denmark; 4μg/ml or 10μg/ml as indicated) or Staphylococcus enterotoxin B (SEB; Sigma–Aldrich, St. Louis, MO; 0.5μg/ml). In some experiments, anti-CD28 and anti-CD49d co-stimulatory antibodies (BD Biosciences, Oxford, UK) were added at 2 μg/ml
Smith S.G., Smits K., Joosten S.A., van Meijgaarden K.E., Satti I., Fletcher H.A., Caccamo N., Dieli F., Mascart F., McShane H., Dockrell H.M, & Ottenhoff T.H. (2015). Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines. PLoS ONE, 10(9), e0138042.
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2

Flow Cytometric Analysis of Antigen-Specific CD4+ T Cells

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The frequency of antigen-specific CD4+ T cells secreting IFN-γ, TNF, and/or IL-2 was detected using flow cytometry31 (link). Briefly, splenocytes were ex vivo stimulated with either the gE OLP pool of total 62 peptides or 0.3% DMSO as a formulation control in the presence of anti-CD28 and anti-CD49d costimulatory antibodies (BD Biosciences), and then incubated for 2 h at 37 °C. Following the antigen stimulation, Brefeldin A (BD Biosciences) was added to splenocytes and further incubated overnight. After stimulation, splenocytes were stained with a mixture of 7-AAD, anti-CD3-FITC, anti-CD8-PE-Cy7, and anti-CD4-V500 (BD Biosciences). For ICS, surface-labeled splenocytes were permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) and labeled with a mixture of anti-TNF-α-PE, anti-IL-2-V450, and anti-IFN-γ-APC (BD Biosciences). Stained samples were analyzed using an LSRII flow cytometer (BD Biosciences) and FlowJo software (FlowJo, LLC).
Nam H.J., Hong S.J., Lee A., Kim J., Lee S., Casper C., Carter D., Reed S.G., Simeon G, & Shin E.C. (2022). An adjuvanted zoster vaccine elicits potent cellular immune responses in mice without QS21. NPJ Vaccines, 7, 45.
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3

Intracellular Cytokine Staining of Whole Blood

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We processed heparinized whole blood for the standardized 12-hour whole blood intracellular cytokine staining (WB-ICS) assay, as previously described(33 (link), 34 (link)) within a maximum of 45 minutes from phlebotomy. Briefly, blood was stimulated with antigens at 37°C for 12 hours. Brefeldin-A (10μg/mL, Sigma Aldrich, St. Louis, USA) was added for the final 5 hours of stimulation. Stimulants included BCG vaccine SSI (1.2 × 106 CFU/mL, Biovac, Cape Town, South Africa), DH5-α E.coli (dose titrated for maximal IFNγ expression by MAIT cells), 15-mer peptide pools spanning ESAT-6/CFP-10 (1μg/mL/peptide, Peptide Protein Research Ltd, London, UK). Phytohaemagglutinin (PHA) was used as a positive control at 5μg/mL, and RPMI only was the negative unstimulated control. ICS assay stimulations were performed in the presence of anti-CD28 and anti-CD49d co-stimulatory antibodies (each at 0.5μg/mL BD BioSciences). At the end of stimulation, blood was chelated with 20μM of EDTA (Sigma-Aldrich) and fixed with 1:10 FACS Lysing solution (BD) to lyse red blood cells.
Suliman S., Murphy M., Musvosvi M., Gela A., Meermeier E.W., Geldenhuys H., Hopley C., Toefy A., Bilek N., Veldsman A., Hanekom W.A., Johnson J.L., Boom W.H., Obermoser G., Huang H., Hatherill M., Lewinsohn D.M., Nemes E, & Scriba T.J. (2019). MR1-Independent Activation of Human Mucosal-Associated Invariant T Cells by Mycobacteria. The Journal of Immunology Author Choice, 203(11), 2917-2927.
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