Name: characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols
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Description: Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

This research is supported by the National Institute on Alcohol Abuse and Alcoholism, award K99/R00 AA021264. Additional lab support as part of startup package has been received from the Department of Immunology, Institute of NeuroImmune Pharmacology, Herbert Wertheim College of Medicine, and FIU- Office of Research and Economic Development. Gianna Casteleiro was supported by NIH/NIGMS R25 GM061347. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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
<ol>
	<li>Perform a 1:1 dilution of blood with 1x-phosphate-buffered saline in a T75 flask.</li>
	<li>Pipette 15 ml of density gradient solution into 50 ml centrifuge tubes and carefully layer (25 - 30 ml/tube) of t...

<ol>
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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...

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 substance abuse research in order to solve unknown mechanisms and pathways such as those associated with alcohol dependence 11-14, drug dependence 13,15, and the combination of HIV infection and substance abuse 16-19. These ongoing studies and future research studies in the field of immunology make in vitro generation of DCs extremely important for research. However, there are several difficulties associated with isolating DCs from human blood as they only constitute 0.1 - 1% of total blood mononuclear cells 20.
To date, some of the well-established methods for the generation of DCs in vitro consists of plastic or glass adherence of monocytes 21,22, density gradient centrifugation 23, specific marker based separation such as magnetic activated cell sorting 22, fluorescent activated cell sorting 24, positive selection of CD14+ monocytes using dextran-coated magnetic nanoparticles 25, and rapid isolation of highly purified monocytes using fully automated negative cell selection 26. However, the best method of choice remains controversial. Therefore, to improve DC generation techniques, several methods have been developed in which the purity of these cells can be greatly increased by differentiation from purified CD34+ progenitor cells and monocytes isolated from peripheral blood mononuclear cells (PBMCs) 27. As mentioned prior, a widely used and popular method for generating monocyte derived dendritic cells (MDDCs) is to explore the ability of monocytes to adhere to glass or plastic (adherence method) 21,22,27. The adherence method is a rapid and straightforward method that does not require the use of complex equipment. However, some disadvantages of this process include lymphocyte contamination, low flexibility, and monocyte transient manipulation 28. An alternative method to the adherence method is the magnetic isolation of monocytes from total PBMCs , particularly with the use of a human monocyte enrichment kit, which is designed to isolate monocytes from PBMCs by negative selection 26. During this procedure, unwanted cells are targeted for removal with tetrameric antibody complexes and dextran-coated magnetic particles. The advantage of this isolation method is that the unwanted labeled cells are separated using a magnet while target cells can be freely poured off into a new tube without the need for columns. To date, with the availability of specific monoclonal antibodies that can label unique cell populations, the magnetic separation technique has become not simply an additional method, but a necessity for the isolation of rare cells in the field of immunology. For instance, techniques such as magnetic cell sorting with commercially available paramagnetic MACS-nanoparticles have facilitated the development of new approaches for research and clinical applications 22,29. Furthermore, recent research studies comparing DC generation from monocyte adherence and MACS technology methods have demonstrated a higher DC purity and viability using MACS separated monocytes 22,30.
The current study presents a comparison between two methods for the generation of human DCs from monocytes isolated from PBMCs: 1) monocyte isolation by adherence and 2) monocyte isolation by negative selection using a commercial human monocyte enrichment kit. This study provides evidence to show that the negative selection magnetic separation procedure to isolate monocytes generates the highest yield of monocytes with no significant differences in monocyte viability when compared with monocytes isolated by adherence method. In turn, after seven days, the monocytes isolated by magnetic separation differentiated into MDDCs with significantly higher proliferative capacity and higher amount of cells expressing double positive (CD11c+/CD14+) phenotype without affecting MDDC viability. Overall, the current study differs from the previous studies referenced above since it demonstrates the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into CD11c+ MDDCs (&#62; 70%) after seven days in culture without compromising their viability. In addition, the current approach provides for the first time characterization of different CD11c/CD14 MDDCs populations by imaging flow cytometry.
In summary, since DCs play a focal role regarding research in the field of immunology, different parameters must be taken into consideration when considering how they are derived and what methods are used to isolate and culture them in vitro. Therefore, this study aims to provide insights on two different methods of monocyte isolation and how these methods differentially affect monocyte viability and yield eventually affecting dendritic cell viability, proliferation, and phenotype. These findings will contribute greatly to the field of immunology and will provide a detailed protocol of DC isolation, purification, and characterization.

<table border="0" cellpadding="0" cellspacing="0" width="1435">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:535px;">Ficoll-Paque</td>
			<td style="width:228px;">GE Healthcare</td>
			<td style="width:231px;">17-5442-03</td>
			<td style="width:443px;">Must be used at room temperature</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phosphate-buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">ACK Lysing buffer</td>
			<td>Quality Biological</td>
			<td>118-156-101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Antibiotic-Antimycotic (100X)</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">RoboSep buffer</td>
			<td>StemCell</td>
			<td>20104</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">EasySep Human monocyte enrichment kit</td>
			<td>StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0101</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FITC-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Trypan blue stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Synergy 2 multi-mode reader</td>
			<td>Biotek</td>
			<td>7131000</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">XTT Sodium salt bioreagent (XTT)</td>
			<td>Sigma Aldrich</td>
			<td>X4626-100MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Dimethyl sulfoxide bioreagent (DMS)</td>
			<td>Sigma Aldrich</td>
			<td>D8418-500ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Thiazolyl blue tetrazolium bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>m5655-500MG</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td>Bio-Rad</td>
			<td>161-0302</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Phenazine Methosulfate (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>P9626-1G</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Inactivated (HI) Human Serum</td>
			<td>Chemicon</td>
			<td>S1-100ML</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Accuri C6 Flow Cytometer</td>
			<td>BD Accuri</td>
			<td>653119</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of human monocyte-derived dendritic cells by imaging flow cytometry: a comparison between two monocyte isolation protocols

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 &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

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...

Watch this Scientific Journal Video about Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols at JoVE.com

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

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.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

The overall goal of this study is to compare two different methods of human monocyte isolation for dendritic cell generation in vitro and to characterize the CT11c, CT14 cell surface expression of the resulting monocyte derived populations by imaging flow cytometry. These monocytes isolation protocols can contribute to the development of reliable methods for the purification of human dendritic cells for research and clinical applications. The main advantage of these protocols is that they facilitate the isolation of human monocytes and their differentiation into viable and functional monocyte derived cells.In addition, this is the first study to characterize the specific population of monocyte derived dendritic cells by single cell imaging flow cytometry. This method can also be applied as an alternative for the isolation of other rare cells at an adequate yield for downstream research applications. Visual demonstration of these methods is critical as it can be challenging to handle the blood samples safely without the appropriate instruction and experience.Begin by diluting the blood sample to a one to one ratio with PBS in a T75 flask. Then, add 15 milliliters of density gradient solution into a 50 milliliter centrifuge tube and carefully layer 25 to 30 milliliters of the diluted blood over the gradient. To achieve a proper separation of the peripheral blood mononuclear cells, load the blood onto the density gradient solution quickly, but carefully and without mixing the layers.Separate the cells by centrifugation, followed by the transfer of the white blood cell interface layer into a new 50 milliliter conical tube. Wash the cells two times in PBS, re-suspending the final pellet in 10 milliliters of ammonium chloride potassium lysing buffer for 15 minutes at 4 degrees celsius. Then wash the cells two more times in PBS.To isolate monocytes by the adherence method, culture five times 10 to the seven cells of the resulting PBMC pellet per 10 milliliters of complete medium in a new T75 flask for two hours at 37 degrees celsius and five percent carbon dioxide in a humidified incubator. At the end of the incubation, discard the non-adherent floating cells and gently wash the adherent cells two times with PBS. Then, feed the adherent cells with complete cell culture medium supplemented with human GMCSF and IL4 and return the culture to the incubator for five to seven days.To isolate the monocytes by magnetic separation, after collecting the PBMC by density gradient separation transfer the white blood cell layer to a five milliliter polystyrene tube and re-suspend the cells in PBS at a five times 10 to seven cells per milliliter concentration. Next, add 50 microliters of a human monocyte enrichment cocktail per milliliter to the cells with mixing and incubate the cells with the cocktail at four degrees celsius for 10 minutes. Then add 50 microliters of magnetic particles per milliliter of cells with careful mixing and incubate the cells at 4 degrees celsius for five minutes.At the end of the second incubation, add PBS to the cells to bring the total volume up to two point five milliliters and mix by pipetting two to three times. Then place the polystyrene tube in an appropriate magnetic device at room temperature. After two and a half minutes, with the tube still in the magnet, decant the purified monocyte fraction into a new 15 milliliter conical tube.Then culture the purified monocytes and complete cell culture medium supplemented with GMCSF and IL4 for five to seven days as just demonstrated. After seven days of differentiation, dispense one times 10 to the six cell aliquats of the monocyte derived dendritic cells into the appropriate number of one point five milliliter tubes, and add 50 microliters of heat inactivated human serum to each sample to block any non-specific finding. After 10 minutes, centrifuge the cells and re-suspend the pellets in the appropriate fluorescence conjugated primary antibodies for 20 minutes at four degrees celsius protected from light.Then wash the cells two times in one milliliter of PBS, resuspending the pellets in 100 microliters of PBS per one times ten to the six cells. Just before the analysis, add one microliter of DAPI to each tube. Then, read the samples on a single cell imaging flow cytometer according to the manufacturer's instructions.On average, monocytes isolated by the adherence method account for approximately six point two percent of the total peripheral blood monocyte cell, or PBMC, population, while the monocytes isolated by magnetic separation account for up to 25 percent of the total PBMC. Monocytes isolated by either method are equally viable. MDDCs differentiated from monocytes purified by both methods were first gated based on DAPI negative or viable cells from total single cells, followed by gating of each cell population.Both isolation methods yielded a low single CD14 positive population and both methods yielded a high total CD11c positive population. Further phenotypic analysis was performed on all the CD11c positive cells and even though magnetic isolation gives a higher percentage of double positive CD11c positive CD14 positive cells, this effect was non-significant when compared with the adherence method. These double positive CD11c positive, CD14 positive cells may be early stage differentiated MDDCs that have not yet dropped the CD14 monocytic marker.When analyzing the single CD11c positive cells, the adherence method gives the highest yield of CD11c positive, CD14 negative cells. These single positive cells may be late stage differentiated MDDCs. Once mastered, the adherence and magnetic isolation techniques can be completed in three to four and one to two hours respectively if they are performed properly.While attempting this procedure, it's important to remember to have fresh dendritic cell differentiation cytokines every 48 hours when the medium is replenished. Working with biohazard fluids such as blood, can be extremely harmful, and the use of appropriate personal protective equipment and disposal methods should always be taken when performing these procedures. This study paves the way for researchers in the field of immunology to explore the use of human monocytes and dendritic cells for therapeutic treatment.After watching this video, you should have a good understanding of how to optimize human monocyte isolation for generating different populations of monocyte derived cells in vitro.

Research

JoVE Journal

Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...