Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes
Published: July 31, 2023
Abstract
Source: Sim, W. J. et al., Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes. J. Vis. Exp. (2016).
This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.
Protocol
All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) Preparation of Reagent Prepare PBS/EDTA: phosphate-buffered saline solution (PBS) and supplement with 2 mM ethylenediaminetetraacetic acid (EDTA). Sterilize this solutio…
Disclosures
The authors have nothing to disclose.
Materials
| Ficoll | GE Healthcare | 17-1440-03 | PBMC isolation |
| Syringe | Becton, Dickinson | 302832 | PBMC isolation |
| 1.5 mL centrifuge tube | Axygen | MCT-150-C | PBMC isolation |
| 15 mL falcon tube | Falcon | 352096 | PBMC isolation |
| 50 mL falcon tube | Falcon | 352070 | PBMC isolation |
| Centrifuge | Eppendorf | 5810R | PBMC isolation |
| 0.2 µm filter | Sartorius stedim biotech | 17597 | PBMC isolation |
| MACs kit | Miltenyi biotec | 130-042-201 | Monocyte enrichment |
| MiniMACS Separator | Miltenyi biotec | 130-042-102 | Monocyte enrichment |
| Cell culture-grade water | Invitrogen, Life Technologies | Cell culture | |
| RPMI | Gibco, Life Technologies | 11875-093 | Cell culture |
| FBS | Hyclone | SH30070103 | Cell culture |
| Penicillin-streptomycin | Gibco, Life Technologies | 15140 | Cell culture |
| Phosphate-Buffered Saline | Gibco, Life Technologies | 10010-031 | Cell culture |
| NEAA | Gibco, Life Technologies | 11140-040 | Cell culture |
| EDTA | Gibco, Life Technologies | 15575 | Cell culture |
| HEPES | Gibco, Life Technologies | 15630-080 | Cell culture |
| Sodium Pyruvate | Gibco, Life Technologies | 11360-070 | Cell culture |
| GM-CSF | Miltenyi biotec | 130-093-868 | Cell culture |
| IL-4 | Miltenyi biotec | 130-093-924 | Cell culture |
| Vitamin D3 | Sigma | D1530 | Cell culture |
| Dexamethasone | Sigma | D2915 | Cell culture |
| LPS | Sigma | l2755 | Cell culture |
| Trypan blue | Gibco, Life Technologies | 15250-061 | Cell culture |
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Cite This Article
Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes. J. Vis. Exp. (Pending Publication), e21522, doi: (2023).