Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Summary

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Abstract

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity.

The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to naïve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Introduction

Dendritic cells (DCs) are the most important specialized antigen-presenting cells of our immune system. Immature DCs (iDCs) reside in the skin or in mucosal tissues and are therefore among the first immune cells to interact with invading pathogens. DCs represent the bridge between the innate and the adaptive immune system¹, since they can activate T- and B-cell responses following pathogen detection. Furthermore, they contribute to pro-inflammatory immune responses because of the secretion of high amounts of cytokines, such as IL-1β, IL-6, and IL-12. DCs also activate NK cells and attract other immune cells to the site of infection by chemotaxis.

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Protocol

Ethics statement: Written informed consent was obtained from all participating blood donors by the Central Institute for Blood Transfusion & Immunological Department, Innsbruck, Austria. The use of anonymized leftover specimens for scientific purposes was approved by the Ethics Committee of the Medical University of Innsbruck.

1. Enrichment of Peripheral Blood Mononuclear Cells (PBMCs)

1. Concentration of PBMCs by centrifugation.
   1. Open the blood pack and split blood in sterile 50 ml centrifuge tubes according to the amount of blood received.
   2. If necessary, adjust the volume to 50 ml for each tube with sterile D....

Results

After centrifugation of anti-coagulated blood using a sucrose cushion, peripheral blood mononuclear cells (PBMCs) are enriched in an interphase on top of the density gradient medium (Figure 1). After the PBMCs are drawn off, FACS analysis is performed to characterize the different cell populations within the PBMCs using lineage markers (e.g., CD3 for T lymphocytes, CD14 for monocytes, and CD19 for B lymphocytes). Figure 2A shows the results of a .......

Discussion

This protocol describes the generation of monocyte-derived dendritic cells (MDDCs) through the isolation of human monocytes from anti-coagulated blood using a magnetic nanoparticle-based assay. In this protocol, the centrifugation steps are performed upstream the cell isolation procedure, which leads to an enrichment of the PBMC fraction. Although cells are lost during centrifugation, to overlay the content of a whole blood pack on density gradient medium would require 200-300 ml of the density gradient medium and theref.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
APC Mouse Anti-Human CD19 Clone HIB19	BD Biosciences	555415
APC Mouse Anti-Human CD83 Clone HB15e	BD Biosciences	551073
BD Imag Anti-Human CD14 Magnetic Particles	BD Biosciences	557769
BD Imagnet	BD Biosciences	552311
BSA (Albumin Fraction V)	Carl Roth	EG-Nr 2923225
Costar 6 Well Clear TC-Treated Multiple Well Plates	Costar	3506
Density gradient media: Ficoll-Paque Premium	GE Healthcare Bio-Sciences	17-5442-03
Dulbecco’s Phosphate Buffered Saline (D-PBS)	Sigma-Aldrich	D8537
Falcon 10 ml Serological Pipet	Corning	357551
Falcon 25 ml Serological Pipet	Corning	357525
Falcon 50 ml High Clarity PP Centrifuge Tube	Corning	352070
Falcon Round-Bottom Tubes	Corning	352054
FITC Mouse Anti-Human CD3 Clone HIT3a	BD Biosciences	555339
Ghost Dye Violet 510 (Cell Viability Reagent)	Tonbo biosciences	13-0870
GM-CSF	MACS Miltenyi Biotec	130-093-862
Heat Inactivated FBS (Fetal Bovine Serum), EU Approved Origin (South America)	Gibco	10500-064
Hettich Rotanta 460R	Hettich	---
IL-4 CC	PromoKine	C-61401
Isolation buffer: BD IMag Buffer (10x)	BD Biosciences	552362
L-Glutamine solution	Sigma-Aldrich	G7513
Microcentrifuge tubes, 1.5 ml, SuperSpin	VWR	211-0015
PE Mouse Anti-Human CD14 Clone M5E2	BD Biosciences	555398
PE Mouse Anti-Human CD209 Clone DCN46	BD Biosciences	551265
RPMI-1640 medium	Sigma-Aldrich	R0883
UltraPure 0.5 M EDTA, pH 8.0	Invitrogen	15575020