Effect of an Immunomodulatory Pharmacological Agent on the Interaction Between Dendritic Cells and CD4+ T Cells
Published: November 30, 2023
Abstract
Source: Wei, H., et al. Development and Functional Characterization of Murine Tolerogenic Dendritic Cells. J. Vis. Exp. (135), (2018).
This video demonstrates a method of converting dendritic cells (DCs) to tolerogenic DCs (TolDCs) using an immunomodulatory pharmacological agent. This conversion is confirmed by evaluating the TolDC function through their immunosuppressive effect on CD4+ T cells.
Protocol
1. Evaluate the Function of TolDCs In Vitro and In Vivo T-cell Syngeneic Proliferation Assay Sterilize all the surgical instruments via autoclaving and perform the experiment in a class II biological safety cabinet with appropriate safety procedures. Prepare MACS buffer using 0.5% bovine serum albumin (BSA) and 2 mM EDTA in 500 ml PBS. Sterilize the buffer by filtering through a 0.2 µm filter. NOTE: Keep t…
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The authors have nothing to disclose.
Materials
| CDDO-DFPA (RTA-408) | Reata Pharmaceuticals | in house synthesis | Cell culture |
| Mouse GM-CSF | Peprotech Inc | 315-03 | BMDC differentiation |
| Pan dendritic cell isolation kit | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| Pan Dendritic Cell Biotin-Antibody Cocktail | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| CD4+ T cell isolation kit | Miltenyi Biotec Inc | 130-104-454 | T cell proliferation assay |
| CFSE | BioLegend | 423801 | T cell proliferation assay |
| FcR Blocking Reagent | Miltenyi Biotec Inc. | 130-100-875 | T cell proliferation assay |
| Anti-Biotin MicroBeads | Miltenyi Biotec Inc. | 130-104-454 | T cell proliferation assay |
| 1 ml syringe | BD Biosciences | 309626 | T cell proliferation assay |
| BSA | Sigma Aldrich Inc | A2058 | T cell proliferation assay |
| EDTA | ThermoFisher Scientific | 15575020 | T cell proliferation assay |
| LS Column | Miltenyi Biotec Inc. | 130-042-401 | T cell proliferation assay |
| Pre-Separation Filter | Miltenyi Biotec Inc | 130-095-823 | T cell proliferation assay |
| Collagenase D | Sigma Aldrich Inc. | 11088858001 | T cell proliferation assay |
| HBSS | ThermoFisher Scientific | 14025076 | T cell proliferation assay |
| Ovalbumin (OVA) peptide 323–329 | Sigma Aldrich Inc. | O1641 | T cell proliferation assay |
