Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Summary

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Abstract

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded > 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

Introduction

Dendritic cells (DCs) are essential mediators of the innate and adaptive immune systems. They function to induce primary immune responses and facilitate the development of immunological memory. These cells are primarily responsible for antigen capture, migration and T cell stimulation and are therefore referred to as professional antigen presenting cells (APCs) ¹.Manipulation of DCs could be utilized across a wide variety of research fields and in the clinical setting to treat different inflammatory diseases such as HIV ^6,7, cancer ⁸, autoimmune diseases ⁹, and allergic responses ¹⁰. DCs are also being used for su....

Protocol

Overall human blood studies have been reviewed and approved by the Institutional Review Board (IRB) of FIU, IRB protocol approval # IRB-13-0440. Human leukopaks were purchased from the community blood bank in Miami, FL.

1. Isolation of PBMCs by Standard Density Gradient Technique

1. Perform a 1:1 dilution of blood with 1x-phosphate-buffered saline in a T75 flask.
2. Pipette 15 ml of density gradient solution into 50 ml centrifuge tubes and carefully layer (25 - 30 ml/tube) of the diluted blood over this gradient.
3. Centrifuge for 20 min at 1,200 x g with acceleration of 1 and deceleration of 0.
4. After centri....

Results

Monocyte Yield by Magnetic Separation is Higher Compared to Monocyte Yield by Adherence Method

Data presented in Figure 1 display PBMC and monocyte cell counts by the trypan blue exclusion method at the day of isolation of PBMCs and separation of monocytes. On average, monocytes isolated by the adherence method accounted for approximately 6.2 percent of total PBMCs while monocytes isolated by ma.......

Discussion

Based on the known difficulties of isolating and generating MDDCs from human blood, the present study aimed to provide a comprehensive comparison of two well-established methods for the generation of MDDCs. The first method compared is a well-established traditional method for generating MDDCs by exploiting the ability of monocytes to adhere to glass or plastic (adherence method) ^21,22,27. The adherence method is fast and cost effective, and does not require the use of complex equipment. However, some disadvan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ficoll-Paque	GE Healthcare	17-5442-03	Must be used at RT
Phosphate-buffered saline (PBS)	Life Technologies	10010-023
ACK Lysing buffer	Quality Biological	118-156-101
RPMI 1640 medium	Life Technologies	22400-089
Antibiotic-Antimycotic (100x)	Life Technologies	15240-062
Fetal Bovine Serum (FBS)	Life Technologies	16000-044
RoboSep buffer	StemCell	20104
EasySep Human monocyte enrichment kit	StemCell	19059
TC20 Automated cell counter	Bio-Rad	145-0101
FITC-Dextran	Sigma Aldrich	FD4-100MG
Trypan blue stain (0.4%)	Life Technologies	15250-061
Synergy 2 multi-mode reader	Biotek	7131000
XTT Sodium salt bioreagent (XTT)	Sigma Aldrich	X4626-100MG
Dimethyl sulfoxide bioreagent (DMS)	Sigma Aldrich	D8418-500ML
Thiazolyl blue tetrazolium bromide (MTT)	Sigma Aldrich	m5655-500MG
Sodium Dodecyl Sulfate (SDS)	Bio-Rad	161-0302
Phenazine Methosulfate (DMSO)	Sigma Aldrich	P9626-1G
Inactivated (HI) Human Serum	Chemicon	S1-100ML
Accuri C6 Flow Cytometer	BD Accuri	653119
FlowSight Amnis Flow Cytometer	EMD Millipore	100300