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.

studi del sangue umano complessivi sono stati esaminati e approvati dal Institutional Review Board (IRB) di FIU, protocollo IRB approvazione # IRB-13-0440. leukopaks umani sono stati acquistati dalla banca del sangue comunità in Miami, FL. 1. Isolamento di PBMC da Tecnica gradiente di densità standard <ol><li> Eseguire una diluizione 1: 1 di sangue con soluzione salina 1x-tampone fosfato in un pallone T75. </li><li> Pipettare 15 ml di soluzione gradiente di densità in 50 ml provette da ...

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Sulla base delle note difficoltà di isolare e generando MDDCs dal sangue umano, il presente studio ha lo scopo di fornire un confronto completo dei due metodi consolidati per la generazione di MDDCs. Il primo metodo rispetto è un metodo tradizionale consolidata per generare MDDCs sfruttando la capacità dei monociti di aderire al vetro o plastica (metodo aderenza) 21,22,27. Il metodo aderenza è veloce ed economica, e non richiede l&#39;uso di attrezzature complesse. Tuttavia, alcuni svantaggi di questo pro...

Le cellule dendritiche (DC) sono mediatori essenziali del sistema immunitario innato e adattativo. La loro funzione di indurre risposte immunitarie primari e facilitare lo sviluppo della memoria immunologica. Queste cellule sono i principali responsabili per la cattura l&#39;antigene, la migrazione e la stimolazione delle cellule T e sono quindi indicati per le cellule presentanti l&#39;antigene come professionali (APC) 1 .Manipulation delle DC potrebbe essere utilizzato in una vasta gamma di settori di ricerca e in ambito clinico per il trattamento di diverse malattie infiammatorie come l&#39;HIV 6,7, il cancro, le malattie autoimmuni 8 9, e le risposte allergiche 10. DC sono anche utilizzati per la ricerca abuso di sostanze al fine di risolvere i meccanismi sconosciuti e percorsi, come quelli associati con la dipendenza da alcol 11-14, tossicodipendenza 13,15, e la combinazione di infezione da HIV e abuso di sostanze 16-19. Questi studi in corso e studi di ricerca future In campo dell&#39;immunologia fare in vitro generazione delle DC estremamente importante per la ricerca. Tuttavia, vi sono diverse difficoltà legate isolare cellule dendritiche dal sangue umano come costituiscono solo lo 0,1 - 1% delle cellule totali mononucleari del sangue 20. Ad oggi, alcune delle tecniche comunemente per la generazione di cellule dendritiche in vitro costituito da plastica o vetro aderenza dei monociti 21,22, densità centrifugazione in gradiente 23, specifica separazione basato marcatore come cellula magnetica attivato smistamento 22, fluorescente cellule attivate classificare 24, selezione positiva dei monociti CD14 + utilizzando nanoparticelle magnetiche rivestite destrano 25, e un rapido isolamento dei monociti altamente purificati mediante selezione cellulare negativa completamente automatizzato 26. Tuttavia, il miglior metodo di scelta rimane controverso. Pertanto, per migliorare le tecniche di generazione di DC, vari metodi sono stati sviluppati in cuila purezza di queste cellule può essere notevolmente aumentata di differenziazione da cellule CD34 + progenitrici purificati e monociti isolati da cellule mononucleate del sangue periferico (PBMC) 27. Come accennato prima, un metodo ampiamente utilizzato e popolare per la generazione di monociti derivati da cellule dendritiche (MDDCs) è quello di esplorare la capacità dei monociti di aderire al vetro o plastica (metodo di aderenza) 21,22,27. Il metodo aderenza è un metodo rapido e semplice che non richiede l&#39;uso di attrezzature complesse. Tuttavia, alcuni svantaggi di questo processo includono contaminazione linfociti, scarsa flessibilità, e monociti manipolazione transitoria 28. Un metodo alternativo al metodo aderenza è l&#39;isolamento magnetico di monociti da PBMC totali, in particolare con l&#39;uso di un kit monociti arricchimento umano, che è progettato per isolare monociti da PBMC da selezione negativa 26. Durante questa procedura, le cellule indesiderate sono mirati per la rimozione con tetramericcomplessi di anticorpi e particelle magnetiche rivestite destrano. Il vantaggio di questo metodo di isolamento è che le cellule marcate indesiderate vengono separati usando un magnete mentre le cellule bersaglio possono essere liberamente versato fuori in un nuovo tubo senza bisogno di colonne. Ad oggi, con la disponibilità di specifici anticorpi monoclonali che possono etichettare popolazioni cellulari unici, la tecnica di separazione magnetica è diventata non semplicemente un ulteriore metodo, ma una necessità per l&#39;isolamento di cellule rare nel campo della immunologia. Per esempio, tecniche come la cellula magnetica ordinamento con disponibili in commercio paramagnetiche MACS-nanoparticelle hanno facilitato lo sviluppo di nuovi approcci per la ricerca e applicazioni cliniche 22,29. Inoltre, studi recenti ricerche che confrontano generazione DC dai metodi monociti aderenza e la tecnologia MACS hanno dimostrato una maggiore purezza DC e la vitalità usando MACS monociti 22,30 separati. I regali studioun confronto tra due metodi per la generazione di cellule dendritiche umane da monociti isolati da PBMC: 1) isolamento monociti da aderenza e 2) isolamento dei monociti di selezione negativa utilizzando un kit commerciale monociti arricchimento umano. Questo studio fornisce la prova per dimostrare che la procedura di selezione separazione magnetica negativa per isolare monociti genera il più alto rendimento dei monociti senza differenze significative nei monociti vitalità se confrontato con monociti isolati con il metodo di aderenza. A sua volta, dopo sette giorni, i monociti isolati da separazione magnetica differenziate in MDDCs con significativamente più alta capacità proliferativa e una maggiore quantità di cellule che esprimono doppio positivo (+ CD11c / CD14 +) fenotipo senza influenzare MDDC vitalità. Nel complesso, questo studio si differenzia dai precedenti studi citati sopra in quanto dimostra la capacità di entrambe le tecniche per generare simultaneamente monociti che sono in grado di proliferare e differenziarsi in CD11c +MDDCs (&gt; 70%) dopo sette giorni di coltura senza compromettere la loro vitalità. Inoltre, l&#39;attuale approccio fornisce per la prima volta la caratterizzazione di differenti CD11c / CD14 popolazioni MDDCs dal flusso di imaging citometria. In sintesi, poiché DC giocano un ruolo centrale riguardante la ricerca nel campo della immunologia, diversi parametri devono essere presi in considerazione quando si considera come sono derivate e quali metodi sono utilizzati per isolare e cultura li in vitro. Pertanto, questo studio mira a fornire approfondimenti su due diversi metodi di isolamento dei monociti e di come questi metodi influenzano in modo differenziale monociti vitalità e la resa alla fine colpisce la vitalità delle cellule dendritiche, la proliferazione, e fenotipo. Questi risultati contribuiranno notevolmente al campo della immunologia e forniranno un protocollo dettagliato di DC isolamento, purificazione e caratterizzazione.

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			<td style="width:443px;">Must be used at room temperature</td>
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			<td height="34" style="height:34px;">Phosphate-buffered saline (PBS)</td>
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			<td>10010-023</td>
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		<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>
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		<tr height="34">
			<td height="34" style="height:34px;">EasySep Human monocyte enrichment kit</td>
			<td>StemCell</td>
			<td>19059</td>
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		<tr height="34">
			<td height="34" style="height:34px;">TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0101</td>
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		<tr height="34">
			<td height="34" style="height:34px;">FITC-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>FD4-100MG</td>
			<td></td>
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		<tr height="34">
			<td height="34" style="height:34px;">Trypan blue stain (0.4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
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			<td height="34" style="height:34px;">Synergy 2 multi-mode reader</td>
			<td>Biotek</td>
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			<td>Sigma Aldrich</td>
			<td>X4626-100MG</td>
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			<td height="34" style="height:34px;">Dimethyl sulfoxide bioreagent (DMS)</td>
			<td>Sigma Aldrich</td>
			<td>D8418-500ML</td>
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		<tr height="34">
			<td height="34" style="height:34px;">Thiazolyl blue tetrazolium bromide (MTT)</td>
			<td>Sigma Aldrich</td>
			<td>m5655-500MG</td>
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			<td height="34" style="height:34px;">Sodium Dodecyl Sulfate (SDS)</td>
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			<td>161-0302</td>
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			<td>Sigma Aldrich</td>
			<td>P9626-1G</td>
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			<td height="34" style="height:34px;">Inactivated (HI) Human Serum</td>
			<td>Chemicon</td>
			<td>S1-100ML</td>
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		<tr height="34">
			<td height="34" style="height:34px;">Accuri C6 Flow Cytometer</td>
			<td>BD Accuri</td>
			<td>653119</td>
			<td></td>
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		<tr height="34">
			<td height="34" style="height:34px;">FlowSight Amnis Flow Cytometer</td>
			<td>EMD Millipore</td>
			<td>100300</td>
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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> Monociti Resa dalla separazione magnetica è superiore rispetto a monociti Resa secondo il metodo Aderenza I dati presentati in Figura 1 mostra PBMC e conta delle cellule monociti dal metodo di esclusione del trypan blue alla data di isolamento delle PBMC e separazione dei monociti. In media, monociti isolati con il metodo aderenza rappresentato circa il 6,2 per cento di PBMC totali, men...

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.

Caratterizzazione delle derivate da monociti cellule dendritiche umane di Citometria a Flusso Imaging: un confronto tra due protocolli di isolamento dei monociti

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

Research

JoVE Journal

Immunology and Infection

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

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1ÃƒÅ½Ã‚Â² in Human Monocyte-derived Dendritic Cells

Attivazione e misurazione dei NLRP3 inflammasome all&#39;attività utilizzando IL-1β in cellule dendritiche umane derivate da monociti

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

Ricerca

Immunologia e infezioni

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

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

Analisi dei Macrofagi Microglia e dei Monociti dal Sistema Nervoso Centrale dalla Citometria del Flusso

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

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

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

Caratterizzazione dei sottoinsiemi di monociti umani da sangue intero flusso Cytometry analisi

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

Quantificazione dell'attività chemiotatica monocite in vivo e caratterizzazione dei macrofagi derivati monociti del sangue

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

L'elettroporazione di siRNA alla modulazione dell'autofagia nell'herpes Simplex Virus Tipo 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

Polarizzazione delle cellule derivate da monociti umani M1 e M2 e analisi con citometria a flusso sull'infezione da tubercolosi del micobatterio

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

Visualizzazione della prossimità indotta da caspasi infiammatorie nei macrofagi derivati da monociti umani

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

Determinazione dell'immunogenicità del vaccino utilizzando cellule dendritiche derivate da monociti bovini

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