Diese Forschungsarbeiten wurden von der National Science Foundation Division of Chemical, Bioengineering, Umwelt und Transport (CBET) Systeme unter award Zahlen CBET-0853780 und CBET-1159344 und die National Institutes of Health, National Institute of Environmental Health Sciences (NIEHS) unterstützt unter Verleihungsnummer 1R43ES022567-01, und dem National Cancer Institute, Cancer Imaging Program unter Verleihungsnummer CA127745-01. Die Lumina IVIS Instrument in dieser Arbeit verwendet wurde, von der US Army Defense University Research Instrumentation Programm erhalten.

1. Zellpräparation <ol><li> Recover ein Fläschchen bioluminescent HEK293 Zellen aus flüssigem Stickstoff gefroren Lager und wachsen sie in Dulbecco modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Rinderserum, 0,01 mM nicht essentiellen Aminosäuren, 1x Antibiotikum-Antimykotikum und 0,01 mM Natriumpyruvat in einem T 75 Zellkulturflasche bei 37 ° C und 5% CO 2. Hinweis: Die Medien und ergänzende Komponenten auf Zelllinien variieren und sollte daher entsprechend gewählt werden. </li><li> Aktualisieren Medium alle 2-3 Tage bis zum Erreichen ~ 80% Konfluenz. Hinweis: Die Anzahl der Zellen benötigt wird auf dem experimentellen Design basieren. Bei Bedarf können weitere Zellen durch Subkultivierung erhalten werden. </li><li> Ernten Sie die Zellen für die Prüfung. <ol><li> Entfernen Sie das Medium und die Zellen mit Phosphat-gepufferter Kochsalzlösung (PBS) durch vorsichtiges Schwenken des Kolbens und dann Verwerfen des PBS verbracht. </li><li> In Trypsin und Inkubation bei 37 ° C für 2 min,oder bis die Zellen vom Kolben abgelöst. </li><li> Sammeln Sie die abgelösten Zellen in PBS und übertragen sie in ein sauberes Zentrifugenröhrchen. Hinweis: Wenn nonadherent Zelllinien, separate Zellen durch Zentrifugation verwendet und einmal mit PBS waschen. </li></ol></li><li> Bestimmen Sie die Gesamtzahl der Zellen mit einem Hämazytometer oder andere Zelle Zählsystem. </li><li> Zentrifuge die Zellsuspension bei 300 xg für 5 min. Überstand verwerfen und die Zellen in frischem, vorgewärmten Medium. </li><li> Seed gleiche Anzahl von Zellen in einzelnen Vertiefungen einer opaken Multi-Well-Platte. In diesem Beispiel Platte 5 x 10 5 Zellen in einem Volumen von 1 ml pro Vertiefung in eine schwarze 24-Well-Platte. Platte ein gleiches Volumen des Mediums in drei Vertiefungen, die als negative Kontrolle dienen. Hinweis: Die Anzahl der Zellen in jeder Vertiefung beschichtet ist flexibel und kann bei einzelnen Versuchen optimiert werden. Für jede Zelllinie Biolumineszenz, experimentell die Beziehung zwischen Zellzahl und bioluminescent Ausgang, sowie die minimal nachweisbare Zelle vor allen Prüfungen der Toxizität zählen. Um diese, messen Biolumineszenz in einem breiten Spektrum von Bevölkerung Größen (z. B. 1 × 10 3 bis 1 x 10 6 Zellen) tun unter standardisierten Bedingungen Bildgebung. </li><li> Gönnen Zellen mit Tests Verbindung (en), wie unten beschrieben. </li></ol> 2. Chemische Vorbereitung <ol><li> Bereiten Sie die Chemikalie als konzentrierte Stammlösung getestet. In diesem Beispiel verwenden Sie eine 100 mg / ml Stammlösung eines Antibiotikums der Bleomycin Familie. Hinweis: Um potenzielle Fahrzeug basierte toxischen Effekte zu minimieren, vor allem, wenn ein organisches Lösungsmittel als Vehikel für chemische Zusatz verwendet wird, empfiehlt es sich, dass die Aktie Konzentration mindestens 1.000 Mal die gewünschte Anwendung post-Konzentration sein. </li><li> In den vorbereiteten chemischen Aktien direkt zu den Zellen. In diesem Beispiel Dosis des Antibiotikums von Interesse in Endkonzentrationen von 100, 200, 300 und 400 pg / ml in triverkomplizieren Brunnen. Lassen Sie drei Brunnen von Zellen unbehandelt als Kontrollen. Hinweis: Die Endkonzentration des Zielchemikalie sollte auf spezielle experimentelle Ziele ausgewählt werden. Eine breite Palette von Konzentrationen getestet, um normalerweise das volle Spektrum der toxischen Effekte zu erzielen. </li></ol> 3. Imaging und Data Analysis <ol><li> Unmittelbar nach Anlagerung wird die Platte in der Imaging-Kammer geeignete Instrument für die Bildaufnahme und Biolumineszenz-Messung. </li><li> Measure Biolumineszenz alle 15 min über einen Zeitraum von 24 Std. mit einem 10 min Integrationszeit für jede Lesung. Hinweis: Die Integrationszeit in diesem Beispiel verwendet wird, ist auf einer Pro-Platte zur Verfügung und können für die Messung auf einer pro-und Basis mit anderen Luminometer der Wahl eingestellt werden. Die Integrationszeit kann auch basierend auf der gewählten Biolumineszenz-Zelllinie eingestellt werden. Weil Biolumineszenz autonom ohne Zell-Zerstörung oder externe Stimulation erzeugt, gelesengen kann an jeder beliebigen Zeitpunkt gemacht werden, um bestimmte Ziele zu erfüllen experimentellen. </li><li> Quantifizierung der Biolumineszenz Intensität aus jeder Vertiefung durch die Identifizierung eines Bereichs von Interesse mit kompatibler Software. Anzeige der Lichtleistung entweder als Gesamtfluß (Photonen / Sekunde) oder der durchschnittlichen Strahlung (Photonen / Sekunde / cm 2 / steridian) jeder Probe. </li></ol>

<ol>
	<li>U.S. Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+really+FDA+approved?.">Is it really FDA approved?.</a> FDA Consumer Health Information. 2, (2009).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+for+the+twenty-first+century.">Toxicology for the twenty-first century.</a> Nature. 460, 208-212 (2009).</li><li>Olson, H., 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=Concordance+of+the+toxicity+of+pharmaceuticals+in+humans+and+in+animals.">Concordance of the toxicity of pharmaceuticals in humans and in animals.</a> Regul. Toxicol. Pharmacol. 32, 56-67 (2000).</li><li>Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+developments+in+cell+culture+technology.">Current developments in cell culture technology.</a> Adv. Exp. Med. Biol. 745, 1-13 (2012).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+human+ADME/Tox+drug+properties+in+drug+discovery.">Screening for human ADME/Tox drug properties in drug discovery.</a> Drug Discov. Today. 6, 357-366 (2001).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+to+evaluate+ADMET+drug+properties.">In vitro approaches to evaluate ADMET drug properties.</a> Curr. Top. Med. Chem. 4, 701-706 (2004).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat. Protoc. 4, 582-591 (2009).</li><li>Thompson, E. M., Nagata, S., Tsuji, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+for+the+luciferase+from+the+marine+ostracod+Vargula+hilgendorfii.">Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 6567-6571 (1989).</li><li>Lupold, S. E., Johnson, T., Chowdhury, W. H., Rodriguez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real+time+Metridia+luciferase+based+non-invasive+reporter+assay+of+mammalian+cell+viability+and+cytotoxicity+via+the+beta-actin+promoter+and+enhancer.">A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the beta-actin promoter and enhancer.</a> PLoS ONE. 7, (2012).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bacterial+bioluminescence.">Molecular biology of bacterial bioluminescence.</a> Microbiol. Rev. 55, 123-142 (1991).</li><li>Close, D. 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=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+of+human+toxicants+in+real+time+using+a+human+optimized+bioluminescent+bacterial+luciferase+gene+cassette+bioreporter.">Remote detection of human toxicants in real time using a human optimized bioluminescent bacterial luciferase gene cassette bioreporter.</a> Proc. SPIE 8371, Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II; and Biometric Technology for Human Identification IX. 837117, (2012).</li><li>Close, D. M., Hahn, R. E., Patterson, S. S., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

DM Close, SA Ripp, und GS Sayler sind Gründer und Inhaber von 490 BioTech, Inc.

Dieses Verfahren zeigt die Verwendung von autonom Biolumineszenz Säugerzellen als ein in vitro-Zytotoxizität Screening-Assay, der lebende Zellen kontinuierlich über ihre Lebensdauer verfolgt werden kann. Dieses Protokoll ist flexibel und kann modifiziert werden, um spezielle experimentelle Bedingungen aufzunehmen, wie erforderlich. So stellte das Experiment hier eignet sich für die Verfolgung von akuten toxischen Wirkungen, kann aber angepasst träge oder langfristigen Auswirkungen durch wiederholtes Bildge...

In den USA benötigen Arzneimittel und andere Produkte für den menschlichen Verzehr bestimmt sind umfangreiche Einschätzung, bevor sie für den Verbraucher von der Food and Drug Administration genehmigt werden. Die finanzielle Belastung für die Durchführung dieser Tests auf den Entwickler 1, das im Wesentlichen die Kosten für neue Verbindung Entwicklung und somit führt zu erhöhten Kosten für den Verbraucher gegeben. Während traditionell viel von dieser Screening tierischen Patienten genutzt hat, um als Stellvertreter für den menschlichen Gastgeber fungieren, hat sich diese zu einer großen finanziellen Belastung werden, mit einem geschätzten $ 2800000000 jährlich aufgewendet auf ADME / Tox (Adsorption, Verteilung, Stoffwechsel, Ausscheidung und Toxizität ) Screening allein 2 und Montage Hinweise darauf, dass Tiermodellen nicht verlässlich vorhersagen humantoxikologischen Antworten 3. Daher wurde in vitro menschliche Zellkultur-basierten Tests Popularität in den letzten zwei Jahrzehnten wegen seiner relativ niedrigen gewonnenKosten, höheren Durchsatz und eine bessere Darstellung von menschlichen Bioverfügbarkeit und Toxikologie 4. Aktuelle Zellkultur-basierte Screening-Methoden beschäftigen Toxizität verschiedenen Endpunkten, wie die Messung der ATP-Spiegel, das Screening der Aktivität von endogen verfügbar zytoplasmatischen Enzyme, die Erforschung der Integrität der Zellmembran, oder die Verfolgung der Ebene der mitochondrialen Aktivität, zu evaluieren zellulären Lebensfähigkeit 5 , 6. Unabhängig von dem Endpunkt gewählt alle diese Verfahren erfordern die Zerstörung der Probe vor der Messung entnommen werden, wodurch nur Erzeugen von Daten in einem einzigen Zeitpunkt. Als Folge müssen eine große Anzahl von Proben vorbereitet zu sein und behandelt parallel zur grundlegenden toxikologischen Studien Kinetik, erneute Zugabe zu den Kosten und Arbeit für neue Verbindung Entwicklung erforderlich. Alternativ Assays mit Luziferase wie Gaussia Luciferase 7, Vargula Luciferase 8 und 9 Metridia Luciferase abgesondertentwickelt worden, die die Beseitigung der Notwendigkeit zur Zelllyse und einen Bruchteil der Medien für die Endpunktbestimmung, werden diese jedoch noch Abtastung zu vorbestimmten Zeitpunkten beschränkt und erfordern die Zugabe von exogenen lichtaktivierenden Substraten. Um den Nachteil der erforderlichen Probe Zerstörung sowie die Kosten für die Beseitigung Substrate zu vermeiden, wurde eine menschliche Zelllinie entwickelt, dass drückt die volle bakterielle Biolumineszenz (lux) Gen-Kassette (luxCDABEfrp), die für die kontinuierliche Überwachung von lebenden Zellen, die ähnlich ist zu ermöglichen fluoreszierende Farbstoff-basierte Live Cell Imaging, aber ohne die zusätzlichen Photon-aktivierenden und mikroskopische Untersuchungsverfahren. Diese Zelllinie kann konstitutiv Erzeugen eines optischen Signals zur kontinuierlichen, direkten Nachweis ohne äußere Stimulation, wodurch die Zerstörung der Probe. Mechanistisch führen die Biolumineszenz-Signal aus diesen Zellen erzeugts, wenn die luxAB gebildete Enzym Luciferase katalysiert die Oxidation einer langkettigen Fettsäure Aldehyd (synthetisiert und von den luxCDE Genprodukte mit endogenen Substraten regeneriert) in Gegenwart von reduziertem Riboflavinphosphat (FMNH 2, die von FMN durch die FRP-Gens zurückgeführt Produkt) und molekularem Sauerstoff 10. Expression des lux-Kassette in der Wirtszelle ermöglicht somit Licht erzeugt und detektiert werden, ohne Zellzerstörung oder exogene Substratzugabe. Ebenso stellt die Interaktion zwischen den Genen und der lux endogen verfügbar FMN und O 2 Cosubstrate, und der Forderung nach Aufrechterhaltung einer Umgebung, die die Umwandlung von FMN zu FMNH 2 unterstützen kann, dass die resultierende Biolumineszenz-Signal kann nur von lebenden nachgewiesen werden , metabolisch aktiven Zellen. Diese Anforderungen wurden bisher ausgenutzt werden, um die lux-bas demonstrierened bioluminescent Ausgang korreliert stark mit zellulären Bevölkerung Größe 11 und dieser toxischen Verbindung Exposition beeinträchtigt autobioluminescent Produktion in einer Dosis-Wirkungs-weise 12. Wir verwenden hier eine bisher dadurch autobioluminescent humane embryonale Nierenzellen (HEK293)-Zelle 11 an die automatisierte toxikologischen Screening eines Antibiotikums aus der Bleomycin Familie mit bekannten DNA-schädigenden Aktivität als ein repräsentatives Beispiel der Anwendung von autobioluminescent Säugetierzellen zur Toxizität validieren demonstrieren.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

Säugetier-Zell-basierten in vitro-Assays wurden weithin als Alternativen zu Tierversuchen für toxikologische Studien eingesetzt wurden dann aber wegen der hohen Geld-und Zeit Kosten parallel Probenvorbereitung, die aufgrund der destruktiven Natur der Glühwürmchen-Luciferase-basierte Screening-Verfahren notwendig sind begrenzt . Dieses Video beschreibt die Verwendung von autonom Biolumineszenz Säugerzellen, die keine die destruktive Addition eines Luciferinsubstrat als kostengünstige und einfache Methode zur Überwachung der zytotoxischen Wirkung einer Verbindung von Interesse. Säugerzellen stabil exprimieren, die vollständige Bakterien Biolumineszenz (luxCDABEfrp) Genkassette autonom erzeugt ein optisches Signal, das Peaks bei 490 nm ohne Zusatz eines teuren und möglicherweise störende Luciferinsubstrat, Anregung durch eine externe Energiequelle oder Zerstörung der Probe, die traditionell durchgeführt während der optischen Bildgebungsverfahrens. Diese Unabhängigkeit von externer Stimulation stellt die Belastung für die Aufrechterhaltung der Biolumineszenz-Reaktion lediglich auf der Zelle, so dass das resultierende Signal nur während des aktiven Metabolismus detektiert. Diese Eigenschaft macht das lux-exprimierenden Zelllinie ein ausgezeichneter Kandidat für die Verwendung als biosentinel gegen zytotoxische Effekte, da Änderungen in Biolumineszenz Produktion anzeigt nachteilige Auswirkungen auf das Zellwachstum und den Stoffwechsel sind. Ebenso ermöglicht die autonome Natur und der Mangel an erforderlichen Probe Zerstörung wiederholte Abbildung der gleichen Probe in Echtzeit während der gesamten Giftstoff Belichtung und können über mehrere Proben in einem der vorhandenen bildgebende Geräte in einer automatisierten Weise durchgeführt werden.

In dieser Studie wurden die Dynamik autobioluminescent HEK293 Zellen kontinuierlich über einen Zeitraum von 24 Std. in Reaktion auf die Antibiotika Exposition (Abbildung 1) überwacht. Die toxischen Wirkungen dieses Antibiotikums, die ein Mitglied der Familie bekannt, Bleomycin lebenden Zellen durch Bindung an DNA und spalten 13 töten ist, wurden über eine Abnahme der Biolumineszenz-Produktion im Vergleich zu unbehandelten Zellen, die direkt visualisiert werden können durch die Pseudocolo...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian Zellen, die das bakterielle Biolumineszenz Genkassette (<em&gt; Lux</em&gt;) Erzeugen Licht autonom. Die resultierenden Biolumineszenz Dynamik bei Exposition gegenüber chemischen Stoffen haben nachgewiesen, dass die Behandlung Auswirkungen auf das Zellwachstum und den Stoffwechsel zu reflektieren, so dass diese Zellen eine kostengünstige, kontinuierliche, Echtzeit-Toxizität Screening-Tool, das leicht für High-Throughput-Automatisierung angepasst werden kann.

Autonom Bioluminescent Säugerzellen für die kontinuierliche und Echtzeit-Überwachung von Zytotoxizität

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

Research

JoVE Journal

Biology

10.0K Aufrufe.  Oak Ridge National Laboratory. Mammalian Zellen, die das bakterielle Biolumineszenz Genkassette ( Lux) Erzeugen Licht autonom. Die resultierenden Biolumineszenz Dynamik bei Exposition gegenüber chemischen Stoffen haben nachgewiesen, dass die Behandlung Auswirkungen auf das Zellwachstum und den Stoffwechsel zu reflektieren, so dass diese Zellen eine kostengünstige, kontinuierliche, Echtzeit-Toxizität Screening-Tool, das leicht für High-Throughput-Automatisierung angepasst werden kann....

Serie: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing und Subkultivierung Säugerzellen

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Forschung

Biologie

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISSION esiRNA für RNAi-Screening in Säugerzellen

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenese und funktionale Analyse von Ionenkanälen heterolog in Säugerzellen exprimierten

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Profiling von Pre-micro RNAs und microRNAs mittels quantitativer real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

Echtzeit-Überwachung von Ligand-Rezeptor-Wechselwirkungen mit Fluorescence Resonance Energy Transfer

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET-Mikroskopie zur Echtzeit-Überwachung der Signalisierung Events in lebenden Zellen mit unimolekularen Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Überwachung Plasmid-Replikation in lebenden Säugerzellen über mehrere Generationen durch Fluoreszenz-Mikroskopie

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Die Messung der Hämsynthese Levels in Säugerzellen

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Sonde-basierte Echtzeit-PCR Ansätze zur quantitativen Messung von microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

In Situ-Überwachung von transient gebildeten molekularen Chaperon-Baugruppen in Bakterien, Hefe und menschlichen Zellen

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...