Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

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) 

1. Preparation of Reagent
   1. Prepare PBS/EDTA: phosphate-buffered saline solution (PBS) and supplement with 2 mM ethylenediaminetetraacetic acid (EDTA). Sterilize this solution by filtration through a 0.2 µm filter. NOTE to store PBS/EDTA at 4 °C and warm to room temperature before use.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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