Mouse CD4+ T cells were sorted from wild-type mice using CD4+T cell microbeads (Miltenyi Biotec), and the purity of CD4+T cells was >95%. The isolated CD4+ T cells labeled with carboxyfluorescein succinimidyl ester (CFSE, 5 mM; Invitrogen) were treated with or without OE-MSCs-Exos in the presence of anti-CD3 and anti-CD28 mAbs (eBioscience) for 72 h. CFSE fluorescence intensity was analyzed to determine the proliferation of CD4+ T cells by flow cytometry.
Anti cd3 and anti cd28 mabs
Manufactured by Thermo Fisher Scientific
Anti-CD3 and anti-CD28 mAbs are monoclonal antibodies targeted against the CD3 and CD28 surface receptors. These antibodies are commonly used in cell culture applications to activate and stimulate T cells.
Automatically generated - may contain errors
Lab products found in correlation
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
Cd4 t cell microbeads, by Miltenyi Biotec (2 mentions)
96 well plate, by Corning (1 mentions)
Facsaria 2, by BD (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Lactate acid, by Merck Group (1 mentions)
Sodium lactate, by Merck Group (1 mentions)
Fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Apc conjugated anti il 2, by BioLegend (1 mentions)
3 protocols using anti cd3 and anti cd28 mabs
1
Exosome-Mediated Regulation of CD4+ T Cell Proliferation
2
Assessing CD4+ T Cell Proliferation
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Mouse CD4+ T cells were sorted from wild-type mice using CD4+T cell microbeads (Miltenyi Biotec). CD4+ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, 5 mM; Invitrogen), and then co-cultured with MDSCs at a ratio of 1:1 in 96-well plates (Costar) in the presence of anti-CD3 and anti-CD28 mAbs (eBioscience) for 3 days. CFSE fluorescence intensity was analyzed to determine the proliferation of CD4+ T cells by flow cytometry.
3
Cytokine Profiling of Lactate-Treated CD4+ T Cells
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Before flow cytometry analysis, CD4 + T cells were incubated with lactate acid, sodium lactate and HCl (all from Sigma-Aldrich), respectively, for 24 hours. For the detection of intracellular cytokines, in the presence of BFA (brefeldin A), the cells were stimulated with 20 ng/mL phorbol 12myristate 13-acetate (PMA) and 1 μM ionomycin (both from Sigma-Aldrich) during the last 6 hours. Cells were fixed and permeabilized intracellularly by a Fixation and Permeabilization Buffer Set (eBioscience). Cells were then stained with allophycocyanin (APC)conjugated anti-IL-2, phycoerythrin (PE)-conjugated anti-IFNγ or PE-conjugated anti-IL-4 mAbs (BioLegend). For surface antigens analysis, cells were stained with PElabelled anti-CD25 and anti-CD69 mAbs (BioLegend), upon stimulation with anti-CD3 and anti-CD28 mAbs (eBioscience) for 24 hours. Flow cytometry was performed on a FACSAria II (BD Biosciences) and data analysis was performed using the FlowJo software.
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