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…

Divulgaciones

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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Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes. J. Vis. Exp. (Pending Publication), e21522, doi: (2023).

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