Name: isolation, transfection, and culture of primary human monocytes
Uploaded: 2019-12-16T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Die Autoren danken dem HIV Clinical/Tumor Biorepository Core für die Bereitstellung von Patientenproben und dem Cellular Immunology Metabolism Core für die Bereitstellung von Durchflusszytometrieanalysen. Dieses Projekt wurde von NIH P20GM121288 und P30GM114732 finanziert.

Alle unten beschriebenen Methoden wurden vom Louisiana State University Health Sciences Center New Orleans Institutional Review Board genehmigt. Das gesamte Blut wurde nach Einholung der Einwilligung in Kenntnis der Sachlage entnommen.
HINWEIS: Das gesamte Verfahren wird unter sterilen Bedingungen in einer Biosicherheitseinrichtung der Stufe 2 (BSL2) durchgeführt, so dass beim Umgang mit biologischen Materialien Vorsicht geboten ist. Insbesondere wird jeder Schritt mit sterilen Techniken unte...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+M1/M2+polarization+dynamically+adapts+to+changes+in+cytokine+microenvironments+in+Cryptococcus+neoformans+infection.">Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection.</a> MBio. 4 (3), e00264 (2013).</li><li>Raggi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Human+Macrophage+M1-M2+Polarization+Balance+by+Hypoxia+and+the+Triggering+Receptor+Expressed+on+Myeloid+Cells-1.">Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1.</a> Frontiers in Immunology. 8, 1097 (2017).</li><li>Van Overmeire, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-CSF+and+GM-CSF+Receptor+Signaling+Differentially+Regulate+Monocyte+Maturation+and+Macrophage+Polarization+in+the+Tumor+Microenvironment.">M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.</a> Cancer Research. 76 (1), 35-42 (2016).</li><li>Vogel, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+macrophage+polarization+in+vitro:+maturation+and+activation+methods+compared.">Human macrophage polarization in vitro: maturation and activation methods compared.</a> Immunobiology. 219 (9), 695-703 (2014).</li><li>Almeida, M., Cordero, M., Almeida, J., Orfao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+subsets+of+peripheral+blood+dendritic+cells+show+distinct+phenotypic+and+functional+abnormalities+in+HIV-1+infection.">Different subsets of peripheral blood dendritic cells show distinct phenotypic and functional abnormalities in HIV-1 infection.</a> AIDS. 19 (3), 261-271 (2005).</li><li>Ciesek, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+TRAIL-dependent+cytotoxicity+of+CD1c-positive+dendritic+cells+in+chronic+hepatitis+C+virus+infection.">Impaired TRAIL-dependent cytotoxicity of CD1c-positive dendritic cells in chronic hepatitis C virus infection.</a> Journal of Viral Hepatitis. 15 (3), 200-211 (2008).</li><li>Granelli-Piperno, A., Golebiowska, A., Trumpfheller, C., Siegal, F. P., Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1-infected+monocyte-derived+dendritic+cells+do+not+undergo+maturation+but+can+elicit+IL-10+production+and+T+cell+regulation.">HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (20), 7669-7674 (2004).</li><li>Hearps, A. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+TLR2+and+TLR4+in+dendritic+cells+in+response+to+HIV+type+1+and+coinfection+with+opportunistic+pathogens.">Up-regulation of TLR2 and TLR4 in dendritic cells in response to HIV type 1 and coinfection with opportunistic pathogens.</a> AIDS Research and Human Retroviruses. 27 (10), 1099-1109 (2011).</li><li>Hernandez, J. C., Latz, E., Urcuqui-Inchima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+induces+the+first+signal+to+activate+the+NLRP3+inflammasome+in+monocyte-derived+macrophages.">HIV-1 induces the first signal to activate the NLRP3 inflammasome in monocyte-derived macrophages.</a> Intervirology. 57 (1), 36-42 (2014).</li><li>Low, H. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR8+regulation+of+LILRA3+in+monocytes+is+abrogated+in+human+immunodeficiency+virus+infection+and+correlates+to+CD4+counts+and+virus+loads.">TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads.</a> Retrovirology. 13, 15 (2016).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Impairment+of+Myeloid+Dendritic+Cells+during+Advanced+Stage+of+HIV-1+Infection:+Role+of+Factors+Regulating+Cytokine+Signaling.">Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling.</a> PLoS ONE. 10 (10), e0140852 (2015).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+negative+regulators+of+cytokine+signaling+during+chronic+HIV+disease+cause+functionally+exhausted+state+of+dendritic+cells.">Increased expression of negative regulators of cytokine signaling during chronic HIV disease cause functionally exhausted state of dendritic cells.</a> Cytokine. 91, 118-123 (2017).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Li, H., Jiang, T., Li, M. Q., Zheng, X. L., Zhao, G. 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J., Cohen, E. A., Xiong, S. D., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA-1236+inhibits+HIV-1+infection+of+monocytes+by+repressing+translation+of+cellular+factor+VprBP.">miRNA-1236 inhibits HIV-1 infection of monocytes by repressing translation of cellular factor VprBP.</a> PLoS ONE. 9 (6), e99535 (2014).</li><li>Riess, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferons+Induce+Expression+of+SAMHD1+in+Monocytes+through+Down-regulation+of+miR-181a+and+miR-30a.">Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.</a> Journal of Biological Chemistry. 292 (1), 264-277 (2017).</li><li>Buchacher, T., Ohradanova-Repic, A., Stockinger, H., Fischer, M. 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Das vorgestellte Protokoll zeigt die Verwendung von Primärzellen von HIV-infizierten Probanden als Modell für die Untersuchung von Monozyten und Makrophagen. HIV+ Patienten, die sich mit cART unterziehen, leben mehrere Jahre mit einer Infektion und können auch andere Co-Infektionen haben, die mit einem geschwächten Immunsystem zusammenhängen. Um die Immunmodulation in Gegenwart einer chronischen HIV-Infektion zu untersuchen, wurden Zellen direkt von Patienten geerntet. Da sich gezeigt hat, dass miRNAs ein...

Eine Infektion mit dem Humanimmundefizienzvirus-1 (HIV-1) verursacht eine schwere Immunfunktionsstörung, die zu opportunistischen Infektionen und erworbenen Immunschwächesyndromen (AIDS) führen kann. Obwohl HIV-infizierte Patienten, die sich cART unterziehen, durch geringe Viruslasten und normale CD4+ T-Zellzahlen gekennzeichnet sind, kann die Funktion des Immunsystems bei diesen Personen beeinträchtigt werden, was zu einer dysfunktionalen Immunantwort führt, die mit einem erhöhten Risiko für die Entwicklung von Krebs verbunden ist1. Die Mechanismen der Immunfunktionsstörung bei HIV-Patienten auf cART bleiben weitgehend unbekannt. Daher ist die Charakterisierung von immunbasierten Immunzellen durch Patienten und die Untersuchung ihrer Biologie und Funktion ein kritischer Bestandteil der aktuellen HIV-Forschung.
Monozyten und Makrophagen sind wichtige Regulatoren von Immunantworten und spielen eine grundlegende Rolle bei der HIV-Infektion2,3,4,5. Heterogen und plastisch können Makrophagen grob in klassisch aktiviert (M1) oder alternativ aktiviert (M2) klassifiziert werden. Während diese allgemeine Klassifizierung bei der Einrichtung experimenteller Bedingungen notwendig ist, kann der Polarisationsstatus von Makrophagen durch eine Vielzahl von Zytokinen6,7,8,9umgekehrt werden. Obwohl mehrere Studien die Auswirkungen einer HIV-Infektion auf Monozyten und dendritische Zellen untersucht haben, sind molekulare Details von monozytenvermittelten Reaktionen weitgehend unbekannt6,7,10,11,12,13,14,15,16,17,18,19. Unter den Faktoren, die an der Immunzellregulation und -funktion beteiligt sind, haben sich gezeigt, dass microRNAs (miRNAs), kurze nicht-kodierende RNAs, die die Genexpression posttranscriptativ regulieren, eine wichtige Rolle im Kontext wichtiger zellulärer Pfade spielen (d. h. Wachstum, Differenzierung, Entwicklung und Apoptose)20. Diese Moleküle wurden als wichtige Regulatoren von Transkriptionsfaktoren beschrieben, die für die Diktat der funktionellen Polarisation von Makrophagen21unerlässlich sind. Die potenzielle Rolle von miRNAs bei Monozyten von HIV-infizierten Personen, die sich cART unterziehen, wurde untersucht, aber Fortschritte auf diesem Gebiet erfordern viel mehr Arbeit22,23,24,25,26. In diesem Beitrag wird eine optimierte Methode zur Transfektion von miRNAs und siRNAs in primäre menschliche Monozyten von HIV-infizierten Patienten und Kontrollen behandelt.
Dieses Protokoll stützt sich auf handelsübliche Reagenzien und Kits, da die Kontinuität des technischen Verfahrens dazu beiträgt, unnötige experimentelle Variablen bei der Arbeit mit klinischen Proben zu vermeiden. Nichtsdestotrotz gibt die Methode wichtige Tipps (d.h. die Anzahl der zellenbeschichteten oder kurzen Inkubation mit serumfreien Medien, um die Haftung der Zellen an der Platte zu fördern). Zusätzlich werden die in diesem Protokoll verwendeten Polarisationsbedingungen aus veröffentlichten Arbeitenabgeleitet 27,28,29.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Stain Buffer</td>
			<td>BD Horizon</td>
			<td>563794</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD14 PerCP</td>
			<td>Invitrogen</td>
			<td>46-0149-42</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD163 BV711</td>
			<td>BD Horizon</td>
			<td>563889</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD209 BV421</td>
			<td>BD Horizon</td>
			<td>564127</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD80 FITC</td>
			<td>BD Horizon</td>
			<td>557226</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD83 APC</td>
			<td>BD Horizon</td>
			<td>551073</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Sep Direct Human Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19669</td>
			<td></td>
		</tr>
		<tr>
			<td>EIF4EBP1 mAb</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>EIF4EBP1 siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-29594</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovin Serum Defined Heat Inactivated</td>
			<td>Hyclone</td>
			<td>SH30070.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>B43618</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH mAb</td>
			<td>Santa Cruz</td>
			<td>SC-47724</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>HuFcR Binding Inhibitor</td>
			<td>eBiosciences</td>
			<td>14-9161-73</td>
			<td>Flow cytometry- blocking buffer</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>B16406</td>
			<td>Software to analyze flow cytometry data</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O55:B5</td>
			<td>Sigma</td>
			<td>L4524</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA microRNA Mimic hsa-miR-146a-5p</td>
			<td>Qiagen</td>
			<td>YM00472124</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION miRNA Negative Control</td>
			<td>Sigma</td>
			<td>HMC0002</td>
			<td>Scrambled miRNA conjugated with a near infrared dye</td>
		</tr>
		<tr>
			<td>Nunc 35mm Cell Culture Dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>R&#38;D Systems</td>
			<td>204-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>R&#38;D Systems</td>
			<td>216-MC-025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-Glutamine</td>
			<td>Corning</td>
			<td>10040CVMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scrambled Control siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-37007</td>
			<td></td>
		</tr>
		<tr>
			<td>Viromer Blue Transfection Reagent Kit</td>
			<td>Lipocalyx</td>
			<td>VB-01LB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1 Cell Proliferation Reagent</td>
			<td>Roche</td>
			<td>5015944001</td>
			<td>Colorimetric assay to assess cell viability</td>
		</tr>
	</tbody>
</table>

isolation, transfection, and culture of primary human monocytes

Trotz der Einführung einer kombinierten antiretroviralen Therapie (cART) Mitte der 1990er Jahre ist das Humanimmundefizienzvirus (HIV) nach wie vor ein großes Gesundheitsproblem. Während die antiretrovirale Therapie die systemische Viruslast effizient senkt und die normale CD4+ T-Zellzahl wiederherstellt, stellt sie kein vollständig funktionierendes Immunsystem wieder her. Ein dysfunktionales Immunsystem bei HIV-infizierten Personen, die sich cART unterziehen, kann durch Immunaktivierung, frühe Alterung von Immunzellen oder anhaltende Entzündungen gekennzeichnet sein. Diese Bedingungen, zusammen mit komorbiden Faktoren im Zusammenhang mit HIV-Infektion, erhöhen die Komplexität der Krankheit, die nicht leicht in Zell- und Tiermodellen reproduziert werden kann. Um die molekularen Ereignisse zu untersuchen, die der Immunfunktionsstörung bei diesen Patienten zugrunde liegen, wird hier ein System zur Kultur und Manipulation menschlicher Primärmonozyten in vitro vorgestellt. Insbesondere ermöglicht das Protokoll die Kultur und Transfektion von primären CD14+ Monozyten, die von HIV-infizierten Personen, die sich cART unterziehen, sowie von HIV-negativen Kontrollen erhalten werden. Die Methode umfasst Isolation, Kultur und Transfektion von Monozyten und monozytenabgeleiteten Makrophagen. Während handelsübliche Kits und Reagenzien eingesetzt werden, bietet das Protokoll wichtige Tipps und optimierte Bedingungen für die erfolgreiche Haftung und Transfektion von Monozyten mit miRNA-Mimik und Inhibitoren sowie mit siRNAs.

Mit dem beschriebenen Verfahren wurden primäre menschliche Monozyten von HIV-infizierten Personen und gesunden Spendern isoliert. Alle hier vorgestellten Daten stammen von HIV+ Probanden, die sich mit niedrigen (<20 Kopien/ml) oder nicht nachweisbaren Viruslasten und normalen CD4+ T-Zellzahlen unter cART befinden. Unmittelbar nach der Isolierung wurden Zellen befleckt und die Durchflusszytometrie durchgeführt, um die Reinheit der Zellpopulationen zu bestätigen. Di...

Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Isolation, Transfection, and Culture of Primary Human Monocytes

Hier wird ein optimiertes Protokoll zur Isolierung, Kultivierung, Transfekung und Unterscheidung menschlicher Primärmonozyten von HIV-infizierten Personen und gesunden Kontrollen vorgestellt.

Isolation, Transfektion und Kultur der primären menschlichen Monozyten

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

HIV-Infektion verursacht Immunfunktionsstörungen auch bei Personen, die sich einer antiretroviralen Therapie ohne nachweisbares Virus unterziehen. Diese Methode ermöglicht die Untersuchung der Monozytenfunktion, die wichtige Regulatoren der Immunantwort sind. Wir haben diese Technik für die Isolierung, Kultur und Transfektion menschlicher Primärmonozyten optimiert.Wir verwenden diese Methode, um die molekularen Mechanismen der Immunfunktionsstörung bei HIV-infizierten Personen zu verstehen, aber es kann auf alle anderen Immunstudien mit Monozyten angewendet werden. Wenn Dies zum ersten Mal mit menschlichem Blut arbeitet, denken Sie daran, die richtigen Biosicherheitsprotokolle zu befolgen und alle Proben als möglicherweise infektiöses Material zu behandeln. Nach der Sammlung von 40 Milliliter frisches menschliches Vollblut in vier 10 Milliliter EDTA Vakuumröhren, verwenden Sie sterile Technik, um das gesamte Blut in einem einzigen 50 Milliliter konischen Propylen-Röhrchen in einem Biosicherheitsschrank zu übertragen.Fügen Sie nach den Anweisungen des Herstellers aus einem ausgewählten menschlichen Monozyten-Isolationskit zwei Milliliter Monozyten-Isolationscocktail aus dem Kit in die Blutröhre und wirbeln die Magnetperlen aus dem Kit für 30 Sekunden. Fügen Sie dem Blut zwei Milliliter Perlen hinzu und verwenden Sie eine 25 Milliliter serologische Pipette, um die Perlen sorgfältig mit dem Blut zu mischen. Nach fünf Minuten bei Raumtemperatur das Blut gleichmäßig auf vier 50-Milliliter-Röhren aufteilen und 30 Milliliter sterile PBS hinzufügen, ergänzt mit einem Millimolaren EDTA zu jeder Röhre.Mischen Sie die Rohre erneut mit einer 25-Milliliter-Serologischen Pipette aus Kunststoff und legen Sie die Rohre in magnetische Halterungen. Nach 10 Minuten verwenden Sie eine Pipette, um den Inhalt aus der Mitte jeder Röhre zu zeichnen, wobei darauf geachtet wird, nicht mehr als einen Milliliter roter Blutkörperchen zu saugen und den Rohrinhalt in eine von vier neuen 50-Milliliter-Röhren zu geben. Fügen Sie 500 zusätzliche Mikroliter der wirbelnden Magnetperlen in jede Röhre und mischen Sie die Zelllösung sanft.Legen Sie die Rohre für fünf Minuten wieder in die Magnethalter, bevor Sie den Inhalt vorsichtig von der Mitte jedes Rohres in eine von vier neuen 50-Milliliter-Röhren übertragen. Wenn alle Zellsuspensionen übertragen sind, legen Sie jedes neue 50-Milliliter-Rohr für fünf Minuten in die Magnethalter, bevor Sie den Rohrinhalt vorsichtig in einen dritten Satz von vier neuen 50-Milliliter-Röhren übertragen. Dann sammeln Sie die Zellen durch Zentrifugation und setzen Sie alle vier Zellpellets in insgesamt 10 Milliliter sterilen PBS zum Zählen wieder auf.Nach dem Zählen die isolierten Monozyten um ein mal 10 bis sechs Milliliter 37 Grad Celsius serumfreies RPMI 1640 Medium, ergänzt mit Antibiotika, wieder aussetzen und einen Milliliter resuspendierten Zellen in 35-Millimeter-Platten in einem Biosicherheitsschrank versiebenen. Legen Sie dann die Platten 30 bis 60 Minuten in den Zellkultur-Inkubator, damit die Zellen an den Brunnenböden haften können. Um Makrophagen mit einem M1-ähnlichen Phänotyp zu grundieren, fügen Sie 100 Mikroliter fetales Rinderserum mit 25 Nanogramm pro Milliliter GM-CSF zu jeder Platte hinzu.Um Makrophagen mit einem M2-ähnlichen Phänotyp zu grundieren, fügen Sie 100 Mikroliter fetales Rind mit 50 Nanogramm pro Milliliter M-CSF zu jeder Platte hinzu. Zur Monozytentransfektion mit microRNA-Mimik, Inhibitoren oder einer kleinen störenden RNA, nach dem Herstellerprotokoll für das Transfektionskit, verdünnen Sie die ausgewählten RNAs im Puffer auf eine Endkonzentration von 1,83 Mikromolaren in 10 Mikrolitern verdünnter Mimik oder Inhibitor pro Transfektion von einem mal 10 auf das volumen der fünften Zellen. Zur Herstellung des Transfektionsreagenz einen Mikroliter des mitgelieferten Polymers in ein 1,5-Milliliter-Mikrozentrifugenrohr geben und sofort 90 Mikroliter des mit dem Kit bereitgestellten Puffers hinzufügen, um insgesamt 91 Mikroliter Reagenz pro Transfektion zu erhalten.Wirbel den Regenten für drei bis fünf Sekunden und Pipette 90 Mikroliter der Transfektionslösung in die Röhre mit 10 Mikroliter verdünnter RNA. Nach dem sanften Mischen die Lösung 15 Minuten bei Raumtemperatur inkubieren, bevor Sie 100 Mikroliter Transfektionskomplex zu einem Bohrkörper der Zellen hinzufügen. Nach vier Stunden bei 37 Grad Celsius das Medium durch drei Milliliter komplettes Medium ersetzen, das den entsprechenden Wachstumsfaktor enthält.Ersetzen Sie am sechsten Tag der Kultur die Kulturüberstandstoffe aus den M1-Kulturen durch drei Milliliter Medium, ergänzt durch fetales Rinderserum, Antibiotika, Lipopolysaccharid und Interferon-Gamma, und ersetzen Sie die Überdräuer aus den M2-Kulturen durch Medium, das mit fetalem Rinderserum, Antibiotika, M-CSF und Interleukin-4 ergänzt wird. Nach 24 Stunden Kultur, waschen Sie jede Platte mit aktivierten Makrophagen zweimal mit frischen PBS pro Wäsche. Für RNA- und Proteinanalysen lysieren Sie die angeschlossenen Zellen direkt in den Platten, um die Entnahme der freigesetzten Zellinhalte für ihre Analyse zu ermöglichen.Für die zytometrische Durchflussanalyse lösen Sie die Zellen mit zwei Millimolaren EDTA in PBS für 10 Minuten bei 37 Grad Celsius, bevor Sie die Zellen vorsichtig von den Plattenböden kratzen, um ihre Sammlung in 1,5 Milliliter Mikrozentrifugenröhren für ihre Analyse zu ermöglichen. Hier werden repräsentative Histogramme von M1-aktivierten Kontrollzellen und patientenabgeleiteten Zellen mit erhöhten Konzentrationen von CD80- und CD83- und M2-aktivierten Zellen mit erhöhtem CD163- und CD209-Spiegel im Vergleich zu nicht aktivierten T0-Zellen gezeigt. Interessanterweise schienen CD80 und CD83 in kontrollabgeleiteten Zellen stärker exprimiert zu sein als in HIV-abgeleiteten Zellen.Die Transfektion von frisch gesammelten Monozyten mit einer gerifflten, nahinfrarot markierten microRNA führt zu einer Wirkungsgrad von mehr als 90 %, die durch Durchflusszytometrie und konfokale Mikroskopie bestimmt wird. Darüber hinaus reduziert die Transfektion die Lebensfähigkeit von patientenabgeleiteten oder Kontrollzellen unabhängig vom Stadium der Zellreifung oder den Transfektionsbedingungen nicht signifikant. Transfektion führt zu einer effektiven Downregulation des Zielgens bei Transfektion mit der spezifischen kleinen störenden RNA sowohl am ersten Tag als auch am vierten Tag nach der Isolation.Darüber hinaus zeigen Zellen, die mit microRNA-Mimik transfiziert wurden, eine 48- bis 72-fache Zunahme der microRNA-Expression über untransfizierte Zellen, während alle Transfektionskontrollen keine nennenswerten Veränderungen aufweisen. Die Anzahl der plattierten Zellen ist überlebenswichtig. Wir empfehlen eine Million Zellen pro 35-Millimeter-Schale.Die Beschichtung in serumfreiem Medium wird auch die Zellanhaftung erheblich verbessern. Zellen, die mit dieser Methode gewonnen werden, können mit Zytokinen oder Wachstumsfaktoren behandelt werden, um differentiale Reaktionen bei Patienten von Interesse und gesunde Kontrollen zu untersuchen.

isolation-transfection-and-culture-of-primary-human-monocytes

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Menschliche T-Lymphozyten-Isolierung, Kultur und Analyse der Migration In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Forschung

Immunologie und Infektion

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generierung von Organotypische Floßkulturen aus primären humanen Keratinozyten

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolierung humaner Monozyten durch Doppel-Zentrifugation und ihre Differenzierung zu Makrophagen in Teflon-beschichteten Zellkultur-Taschen

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Hocheffiziente Transfektion von humanen THP-1 Makrophagen durch Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymphocyte Isolierung von menschlicher Haut für phänotypische Analyse und<em&gt; Ex Vivo</em&gt; Zellkultur

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Live-Imaging von Anti-Pilz-Aktivität von Human Primary Neutrophilen und Monozyten in Reaktion auf<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Kleine RNA Transfektion in primären humanen Th17-Zellen durch Next Generation Elektroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

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 their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

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

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

Abgeleitet von Maus und Mensch primäre gastrische Epithelzellen Monolayer Kultur for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...