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

Des études globales de sang humain ont été examinés et approuvés par le comité d&#39;examen institutionnel (IRB) de CRF, protocole IRB approbation # IRB-13-0440. leukopaks humains ont été achetés auprès de la banque de sang de la communauté à Miami, Floride. 1. Isolement des PBMC par gradient de densité standard Technique <ol><li> Effectuer une dilution 1: 1 de sang avec une solution saline 1x-tamponnée au phosphate dans un flacon T75. </li><li> Introduire à la pipette 15 ml...

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Sur la base des difficultés connues d&#39;isolement et de générer MDDCs de sang humain, la présente étude vise à fournir une comparaison détaillée des deux méthodes bien établies pour la génération de MDDCs. La première méthode comparée est une méthode traditionnelle bien établie pour générer MDDCs en exploitant la capacité des monocytes à adhérer au verre ou en plastique (méthode d&#39;adhérence) 21,22,27. La méthode d&#39;adhésion est rapide et rentable, et ne nécessite pas l&#39;...

Les cellules dendritiques (CD) sont des médiateurs essentiels des systèmes immunitaires innées et adaptatives. Ils fonctionnent à induire des réponses immunitaires primaires et faciliter le développement de la mémoire immunologique. Ces cellules sont principalement responsables de capture d&#39; antigène, la migration et la stimulation des cellules T et sont donc appelées cellules comme professionnels présentant l&#39;antigène (CPA) 1 .Manipulation des PED pourraient être utilisés dans un large éventail de domaines de recherche et dans le cadre clinique pour traiter différents des maladies inflammatoires telles que le VIH 6,7, le cancer, les maladies auto - immunes 8, 9 et 10 des réactions allergiques. DC sont également utilisés pour la recherche sur la toxicomanie afin de résoudre des mécanismes et des voies inconnues , tels que ceux associés à la dépendance à l&#39;alcool 11-14, la dépendance aux drogues 13,15, et la combinaison de l&#39; infection et de la substance VIH abus 16-19. Ces études en cours et les futures études de recherche in le domaine de l&#39; immunologie faire dans la production in vitro de DC extrêmement important pour la recherche. Cependant, il existe plusieurs difficultés associées à l&#39; isolement DCs à partir de sang humain comme elles ne constituent que 0,1 à 1% du nombre total de cellules mononucléées du sang 20. À ce jour, quelques - unes des méthodes bien établies pour la production de CD in vitro consiste en plastique ou en verre adhérence des monocytes 21,22, la densité centrifugation gradient 23, séparation spécifique à base de marqueur tel que magnétique tri cellulaire activé 22, fluorescent tri cellulaire activé 24, sélection positive des monocytes CD14 + à l&#39; aide de nanoparticules magnétiques dextran revêtues 25, et l&#39; isolement rapide des monocytes hautement purifiés à l&#39; aide de la sélection de cellule négative entièrement automatisé 26. Cependant, la meilleure méthode de choix reste controversé. Par conséquent, pour améliorer les techniques de génération de courant continu, plusieurs méthodes ont été mises au point, dans lequella pureté de ces cellules peut être fortement augmentée par la différenciation de cellules progénitrices CD34 + et les monocytes purifiés isolés à partir de cellules mononucléaires du sang périphérique (PBMC) 27. Comme mentionné avant, une méthode largement utilisée et populaire pour générer monocytes des cellules dendritiques dérivées (de MDDCs) est d&#39;explorer la capacité des monocytes à adhérer au verre ou en plastique (méthode d&#39;adhérence) 21,22,27. Le procédé d&#39;adhérence est une méthode simple et rapide qui ne nécessite pas l&#39;utilisation d&#39;un équipement complexe. Cependant, certains inconvénients de ce procédé comprennent la contamination des lymphocytes, une faible flexibilité et de manipulation monocyte transitoire 28. Une méthode alternative à la méthode d&#39;adhérence est l&#39;isolation magnétique des monocytes à partir de PBMC totaux, en particulier avec l&#39;utilisation d&#39;un kit d&#39;enrichissement de monocyte humain, qui est destinée à isoler des monocytes à partir des PBMC par sélection négative 26. Au cours de cette procédure, les cellules indésirables sont ciblées pour l&#39;élimination de tétramèreles complexes d&#39;anticorps et des particules magnétiques revêtues de dextrane. L&#39;avantage de cette méthode d&#39;isolement est que les cellules marquées non désirées sont séparés à l&#39;aide d&#39;un aimant tandis que les cellules cibles peut être versé librement au loin dans un nouveau tube sans qu&#39;il soit nécessaire pour les colonnes. A ce jour, la disponibilité d&#39;anticorps monoclonaux spécifiques qui peuvent étiqueter des populations de cellules uniques, la technique de séparation magnétique est devenue non seulement une méthode supplémentaire, mais une nécessité pour l&#39;isolement de cellules rares dans le domaine de l&#39;immunologie. Par exemple, des techniques telles que la cellule magnétique de tri disponibles dans le commerce avec paramagnétiques MACS-nanoparticules ont facilité le développement de nouvelles approches pour la recherche et les applications cliniques 22,29. En outre, des études récentes comparant génération DC à partir de méthodes monocytes d&#39;adhérence et de la technologie MACS ont démontré une pureté et la viabilité plus élevée DC en utilisant MACS monocytes 22,30 séparés. Les cadeaux de l&#39;étude en coursune comparaison entre les deux procédés pour la production de CD humaines à partir de monocytes isolés à partir de PBMC: 1) l&#39;isolement de monocytes par adhérence et 2) l&#39;isolement de monocytes par sélection négative en utilisant un kit commercial d&#39;enrichissement de monocytes humains. Cette étude fournit des preuves pour montrer que la procédure de sélection de séparation magnétique négatif pour isoler les monocytes génère le plus haut rendement de monocytes avec aucune différence significative dans la viabilité des monocytes par rapport aux monocytes isolés par la méthode d&#39;adhérence. À son tour, au bout de sept jours, les monocytes isolés par séparation magnétique différenciées en MDDCs avec une capacité proliférative significativement plus élevée et plus grande quantité de cellules exprimant positive double (CD11c + / CD14 +) phénotype sans affecter CDMD viabilité. Dans l&#39;ensemble, l&#39;étude actuelle diffère des études antérieures mentionnées ci-dessus car elle démontre la capacité des deux techniques pour générer simultanément des monocytes qui sont capables de proliférer et de se différencier en cd11cMDDCs (&gt; 70%) après sept jours de culture sans compromettre leur viabilité. En outre, l&#39;approche actuelle prévoit la première caractérisation temporelle de CD11c différents / populations CD14 MDDCs par flux d&#39;imagerie cytométrie. En résumé, étant donné que les PED jouent un rôle central en ce qui concerne la recherche dans le domaine de l&#39; immunologie, des paramètres différents doivent être pris en considération lors de l&#39; examen comment ils sont dérivés et quelles méthodes sont utilisées pour isoler et les cultiver in vitro. Par conséquent, cette étude vise à fournir des indications sur deux méthodes différentes d&#39;isolement des monocytes et comment ces méthodes affectent différemment monocytes la viabilité et le rendement peut éventuellement affecter la viabilité des cellules dendritiques, la prolifération et le phénotype. Ces résultats contribueront grandement au domaine de l&#39;immunologie et fournira un protocole détaillé de DC isolement, la purification et la caractérisation.

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			<td>653119</td>
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			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
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		</tr>
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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.

<html> Monocyte Rendement par séparation magnétique est plus élevé par rapport à monocytes Rendement par adhésion Méthode Les données présentées sur la Figure 1 affichage CMSP et le nombre de cellules de monocytes par la méthode d&#39;exclusion au bleu Trypan à jour de l&#39; isolement des CMSP et la séparation des monocytes. En moyenne, les monocytes isolés par l...

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.

Caractérisation des monocytes dérivées des cellules dendritiques humaines par imagerie cytométrie de flux: une comparaison entre deux protocoles monocytes d&#39;isolement

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

Activation et évaluation de NLRP3 Inflammasome activité de l&#39;IL-1β Utilisation dans des cellules dendritiques humaines dérivées de monocytes

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

Recherche

Immunologie et infection

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Génération de cellules humaines dérivées de monocytes dendritiques du sang total

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

Analyse de la microglie et des macrophages dérivés de monocytes du système nerveux central par cytométrie de flux

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

Isolement et la Culture In Vitro de Macrophages alvéolaires murines et humaines

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

Caractérisation des sous-ensembles de Monocyte humain de sang Flow Cytometry analyse

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

Quantification de l'activité chimiotactique monocyte in Vivo et caractérisation des macrophages dérivés du monocyte sanguin

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

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells SiRNA Electroporation to Modulate Autophagie in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

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

Polarisation des cellules et analyses dérivées des monocytes humains M1 et M2 avec cytométrie du débit sur l’infection par mycobactérie tuberculeuse

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

Visualisation de la proximité induite par les caspases inflammatoires dans les macrophages dérivés des monocytes humains

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

Détermination de l’immunogénicité du vaccin à l’aide de cellules dendritiques dérivées de monocytes bovins

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