Name: flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)
Uploaded: 2017-12-13T14:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

Wir möchte Cairn-Forschung danken für die Arbeit mit uns dieses System vor allem Mark Henson, Jeremy Graham und Joao Correia zu entwickeln. Wir danken auch David Welsh und Akhilesh Reddy für wertvolle Diskussion während der Entwurfsphase, sowie Peter Laskey (früher von Hamamatsu) für die Vermittlung des Darlehens eines Demo-Kamera und David Wong für seine kritische Eingabe des Manuskriptes.

<ol>
	<li>Morgan, L. W., Greene, A. V., Bell-Pedersen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+and+light-induced+expression+of+luciferase+in+Neurospora+crassa.">Circadian and light-induced expression of luciferase in Neurospora crassa.</a> Fungal Genet. and Biol. 38 (3), 327-332 (2003).</li><li>Millar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Circadian+Phenotype+Based+on+Firefly+Luciferase+Expression+in+Transgenic+Plants.">A Novel Circadian Phenotype Based on Firefly Luciferase Expression in Transgenic Plants.</a> Plant Cell Online. 4 (9), 1075-1087 (1992).</li><li>Yu, W., Hardin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Firefly+Luciferase+Activity+Assays+to+Monitor+Circadian+Molecular+Rhythms+In+Vivo+and+In+Vitro.">Use of Firefly Luciferase Activity Assays to Monitor Circadian Molecular Rhythms In Vivo and In Vitro.</a> Circadian Rhythms: Methods and Protocols. , 465-480 (2007).</li><li>Yoo, S. -. 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=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc Natl Acad Sci. 101 (15), 5339-5346 (2004).</li><li>Buhr, D. E., Yoo, S. -. H., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+as+a+universal+resetting+cue+for+mammalian+circadian+oscillators.">Temperature as a universal resetting cue for mammalian circadian oscillators.</a> Science. 330 (6002), 379-385 (2010).</li><li>Brown, S. A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythms+of+mammalian+body+temperature+can+sustain+peripheral+circadian+clocks.">Rhythms of mammalian body temperature can sustain peripheral circadian clocks.</a> Current Biology. 12 (18), 1574-1583 (2002).</li><li>Balsalobre, A., 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=Resetting+of+circadian+time+in+peripheral+tissues+by+glucocorticoid+signaling.">Resetting of circadian time in peripheral tissues by glucocorticoid signaling.</a> Science. 289 (5488), 2344-2347 (2000).</li><li>Balsalobre, A., Marcacci, L., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+pathways+elicit+circadian+gene+expression+in+cultured+Rat-1+fibroblasts.">Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts.</a> Curr. Biol. CB. 10 (20), 1291-1294 (2000).</li><li>Hastings, M. H., Reddy, A. B., McMahon, D. G., Maywood, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circadian+mechanisms+in+the+suprachiasmatic+nucleus+by+transgenesis+and+biolistic+transfection.">Analysis of circadian mechanisms in the suprachiasmatic nucleus by transgenesis and biolistic transfection.</a> Methods Enzymol. 393, 579-592 (2005).</li><li>Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Ba Barres, ., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NS21:+re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: re-defined and modified supplement B27 for neuronal cultures.</a> J. Neurosci Methods. 171 (2), 239-247 (2008).</li><li>Feeney, K. A., Putker, M., Brancaccio, M., ONeill, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+Characterization+of+Firefly+Luciferase+as+a+Reporter+of+Circadian+Gene+Expression+in+Mammalian+Cells.">In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.</a> J. Biol. Rhythms. 31 (6), 540-550 (2016).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Hirota, T., Lewis, W. G., Liu, A. C., Lee, J. W., Schultz, P. G., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+reveals+period+shortening+of+the+mammalian+circadian+clock+by+specific+inhibition+of+GSK-3beta.">A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta.</a> Proc Natl Acad Sci. 105 (52), 20746-20751 (2008).</li><li>Meng, Q., Maywood, E. S., Bechtold, D. A., Lu, W., Li, J., Gibbs, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrainment+of+disrupted+circadian+behavior+through+inhibition+of+casein+kinase+1+(+CK1+)+enzymes.">Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 ( CK1 ) enzymes.</a> Proc Natl Acad Sci. 1, 1-6 (2010).</li><li>Khabirova, E., Chen, K. -. F., O'Neill, J. S., Crowther, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flyglow:+Single-fly+observations+of+simultaneous+molecular+and+behavioural+circadian+oscillations+in+controls+and+an+Alzheimer's+model.">Flyglow: Single-fly observations of simultaneous molecular and behavioural circadian oscillations in controls and an Alzheimer's model.</a> Sci. Rep. 6, 33759 (2016).</li></ol>

Keine Interessenkonflikte bestehen.

Das hier beschriebene Protokoll ist für Säugetier-Zellkultur, beide perfundierten und statischen Bedingungen. Der ALLIGATOR kann jedoch leicht auf andere Modellsysteme angepasst. In der Tat hat sich bereits gezeigt, eine hervorragende Plattform bieten für die gleichzeitige Überwachung der Fortbewegung, Schlaf und peripheren gen Ausdruck Rhythmen in Drosophila Melanogaster unter ständige Dunkelheit15. aufrechterhalten Es wird darauf hingewiesen, dass je nach Anwendung, Kamera-Typen als die hier erwähnten geeignet sein können. Wir uns vorstellen, dass mit den entsprechenden Filtern, eine modifizierte Version des aktuellen Setups im Prinzip für die Fluoreszenz-Quantifizierung verwendet werden könnte.
Die einzigen Anwendungen, für die der ALLIGATOR möglicherweise nicht geeignet, sind diejenigen, für die besonders hoher räumlicher Auflösung erforderlich, z. B. Bildgebung der räumlich-zeitliche Organisation der PER2::LUC Ausdruck in den Säugetieren organotypischen Scheiben suprachiasmatischen Kern, oder andere kleine Gewebe-Scheiben.
Der ALLIGATOR ermöglicht viele Experimente durchgeführt werden, die bisher nicht durch konventionelle Aufnahmetechniken leicht erreichbar gewesen. Im Vergleich mit aktuellen Methoden zur Messung der Biolumineszenz, bietet der ALLIGATOR mehr Flexibilität bei der sowohl die Art der Zelle Kulturschale oder Folie, die verwendet werden kann, die externen Medien Bedingungen, Empfindlichkeit und Processivity.
Dies gilt insbesondere in einer Zeit wenn es eine Abkehr von der standard 2D Kultur Zellmodelle in Richtung 3D organoide und Flow-Kultur-Systeme ist. Als solche wird davon ausgegangen, dass der ALLIGATOR eine anpassungsfähige Methode zur Verfügung stellt, mit der Biolumineszenz über viele Tage und Wochen unter den unterschiedlichsten Bedingungen gemessen werden kann.

Die Verwendung von Luciferases als Reporter der Genexpression und Proteinaktivität ist eine populäre Technik in der molekularen und zellulären Biologie Forschung geworden. Dies gilt im circadianen Bereich, wo die Kinetik der Firefly Luciferase Synthese und katalytische Inaktivierung sind besonders gut geeignet zur Berichterstattung der longitudinalen Veränderungen der Genexpression, die über die ca. 24 h circadiane Zyklus auftreten. Als solche wird Luciferase als circadiane Reporter in verschiedensten Organismen, einschließlich Pilze, Pflanzen, fliegen und Säugetiere1,2,3,4eingesetzt.
Wenn zirkadiane Gene Expression in Vitrozu quantifizieren, ist ein Photomultiplier Schlauch (PMT) verbreitet die biolumineszente Signal aufzeichnen. PMT-basierten Messungen haben jedoch eingeschränkt in der Regel auf einer vorher festgelegten Platte oder Schüssel Größe beschränkt. Es ist auch nicht möglich, alle räumlichen Informationen zu sammeln, aus Proben mit einem PMT, die führen kann zu einem Verlust von Informationen beim Proben abbilden, die räumliche Variation in Luciferase Ausdruck zeigen überwacht. Darüber hinaus wie die PMT und zugehörigen Elektronik anfällig für Fehlfunktionen bei der feuchte Umgebung eine Standardzelle Kultur Inkubator ausgesetzt sind, erfolgt längs Luciferase-Aufnahme mit PMTs stets in Inkubatoren nicht befeuchtet. In der Folge Zelle Kultur Gerichte müssen dicht sein Luft-um Feuchtigkeitsverlust durch Verdunstung zu verhindern und die Nährmedien müssen daher mit 3 - gepuffert werden (N- Morpholino) Propanesulfonic Säure (MOPS) oder 4-(2-Hydroxyethyl)-1- Piperazineethanesulfonic Säure (HEPES), anstatt die CO2/bicarbonate Pufferung System, das funktioniert in Vivo und wird routinemäßig in Säugetieren Gewebekultur verwendet.
Aufgrund dieser Beschränkungen schränkt Messung der Biolumineszenz von PMTs in der Regel engen auf die Bedingungen, unter denen Zellen während der Experimente gewahrt sind. Um diese Probleme zu überwinden, und auch zur Erhöhung der Reichweite der experimentellen Bedingungen, verwenden wir einen standard CO2/n2 170 L Gewebekultur-Inkubator, der durch die Zugabe von einem Wasser-gekühlten Elektron-Multiplikation angepasst wurde – Coupled Ladegerät (EMCCD) Kamera mit Antibeschlag-Optik und die digitale Steuerung von Temperatur und Gas. Dies ist eine automatisierte längs Luciferase Imaging Gas- und Temperature-Optimized Recorder oder ALLIGATOR bezeichnet worden. Der ALLIGATOR ermöglicht eine wesentlich höhere Flexibilität der Biolumineszenz imaging, sowohl für Hochdurchsatz-Bildgebung der standard Gewebekultur Platten (bis zu 6 X 96 oder 384-Well-Platten gleichzeitig) und auch für nicht standardmäßige Gewebekultur-Systeme, so als perfundierten Zellen in mikrofluidischen Geräte angebaut. Dieses Instrument ermöglicht auch imaging zu feuchte Bedingungen und Variable Steuerung der CO2 und O2 Partialdruck sowie Temperatur auftreten.
Das folgende Protokoll beschreibt eine Methode für die Biolumineszenz Aufzeichnung von Säugetierzellen und Gewebekultur Systeme mit einem ALLIGATOR (im folgenden als "Biolumineszenz Inkubator" bezeichnet). Es sei jedoch darauf hingewiesen, dass das System gut geeignet zur Biolumineszenz Bildgebung und auch mit einigen Änderungen, Leuchtstofflampen, imaging, in einer Reihe von anderen biologischen Systemen und Kontexte wäre.

<table><tbody><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>Hyclone FetalClone III Serum</td><td>GE Healthcare</td><td>SH30109.03</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher</td><td>21103049</td><td>basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A4919</td><td></td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I1882</td><td></td></tr><tr><td>Superoxide Dismutase</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>Holo-transferrin</td><td>Calbiochem</td><td>616424</td><td></td></tr><tr><td>T3 (triiodo-L-thyronine</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>L-Carnitine</td><td>Sigma</td><td>C7518</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E9508</td><td></td></tr><tr><td>D (+)-Galactose</td><td>Sigma</td><td>G0625</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>Lipoic Acid</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Retinol Acetate</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Retinol, all trans</td><td>Sigma</td><td>95144</td><td></td></tr><tr><td>D,L-alpha-Tocopherol</td><td>Sigma</td><td>95240</td><td></td></tr><tr><td>D,L-alpha-Tocopherol acetate</td><td>Sigma</td><td>T3001</td><td></td></tr><tr><td>Sodium Bicarbonate Solution</td><td>Sigma</td><td>S8761-100ML</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Galaxy 170R incubator&amp;nbsp;</td><td>Eppendorf</td><td>CO17301001</td><td></td></tr><tr><td>Luciferin</td><td>Biosynth</td><td>L-8220</td><td></td></tr><tr><td>D -(+)-Glucose solution</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>DMEM powder</td><td>Sigma</td><td>D5030</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>PHG0007</td><td></td></tr><tr><td>1 mm I.D. silicone tubing&amp;nbsp;</td><td>GE Healthcare</td><td>19-4692-01</td><td></td></tr><tr><td>Elbow luer connector</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Male luer fittings</td><td>Ibidi</td><td>10826</td><td></td></tr><tr><td>Female luer fittings</td><td>Ibidi</td><td>10825</td><td></td></tr><tr><td>&amp;micro;-slide luer I 0.6</td><td>Ibidi</td><td>80196</td><td></td></tr><tr><td>BD plastipak 20ml syringe</td><td>Becton Dickinson</td><td>300613</td><td></td></tr><tr><td>1mm I.D. ETFE tubing</td><td>GE Healthcare</td><td>18-1142-38</td><td></td></tr><tr><td>PF670462</td><td>Sigma</td><td>SML0795</td><td></td></tr><tr><td>B27 Supplement (50x)</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>iXon Ultra EMCCD camera</td><td>Andor</td><td>iXon 888</td><td></td></tr><tr><td>Fiji</td><td>ImageJ</td><td>N/A</td><td></td></tr><tr><td>Prism 7.0</td><td>Graphpad Software</td><td>N/A</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Deltaphase Isothermal Pad</td><td>Braintree Scientific</td><td>39DP</td><td></td></tr><tr><td>Heated neutral density filter&amp;nbsp;</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>Osmomat 030</td><td>Gonotech</td><td>Discontinued</td><td></td></tr><tr><td>300 mOsmol/kg calibration standard</td><td>Gonotech</td><td>30.9.0020</td><td></td></tr><tr><td>Measuring vessel</td><td>Gonotech</td><td>30.9.0010</td><td></td></tr><tr><td>Focusing cylinder</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>NE-1600 programmable syringe pump</td><td>Pump Systems inc.</td><td>NE-1600</td><td></td></tr><tr><td>Andor Solis Software</td><td>Andor</td><td>N/A</td><td></td></tr></tbody></table>

flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)

Luciferase-basierte Reporter der zellulären Genexpression sind weit verbreitet für beide längs- und Endpunkt assays biologischer Aktivität. In der zirkadianen Rhythmen Forschung entstehen z. B. Uhr gen Fusionen mit Firefly Luciferase robuste Rhythmen in zellulären Biolumineszenz, die über viele Tage hinweg beibehalten. Technische Einschränkungen verbunden mit Photomultiplier Tubes (PMT) oder konventionellen Mikroskopie-basierte Methoden zur Quantifizierung der Biolumineszenz haben in der Regel gefordert, dass Zellen und Gewebe ganz physiologischen Bedingungen während beibehalten werden Aufnahme mit einem Kompromiss zwischen Empfindlichkeit und Durchsatz. Hier berichten wir über eine Verfeinerung der vorherigen Methoden, die ermöglicht langfristige Biolumineszenz Bildgebung mit hoher Empfindlichkeit und Durchsatz die unterstützt eine breite Palette von Kulturbedingungen, einschließlich variabler Gas und Feuchtigkeitskontrolle, und akzeptiert, dass viele verschiedene Gewebekultur und Geschirr. Diese automatisierte längs Luciferase imaging-Gas - und Temperatur-optimierte Recorder (ALLIGATOR) auch ermöglicht die Beobachtung von räumlichen Variationen in Luciferase Ausdruck über eine Zelle Monolage oder Gewebe, das leicht durch herkömmliche beobachtet werden können Methoden. Wir heben hervor, wie der ALLIGATOR erheblich erhöhte Flexibilität für die Erkennung der Luciferase-Aktivität im Vergleich mit bisherigen Verfahren bietet.

Dieser Artikel beschreibt ein Protokoll für die Biolumineszenz Bildgebung von Säugerzellen mit einem ALLIGATOR (Biolumineszenz Brutkasten). Diese Technik ermöglicht eine Flexibilität der physische Aufbau und extrazelluläre Bedingungen beim biolumineszente Systeme abbilden. Methoden für einfache statische Gewebekultur (Abbildung 1, ergänzende Video 1) und perfundierten Zellkultur (Abbildung 2, ergänzende Video 2) beschrieben, aber viele andere Setups konnte mit diesem System abgebildet werden. Alle Daten wurden mit Hilfe der in Abschnitt 7 beschriebenen Methoden quantifiziert.
Abbildung 1A zeigt ein Beispiel video von einer Aufnahme von 6 x 96-Well-Platten mit PER2::LUC Fibroblasten4Biolumineszenz. Die Regionen in äußerster Brunnen enthalten keine Zellen, da diese nicht erforderlich für dieses Experiment. Zellen haben Differenztemperatur Entrainment, wobei sie entweder Temperatur-Zyklus von 12 h, bei 32 ° C, gefolgt von 12 h 37 ° C für 72 h oder das Gegenteil (12 h bei 37 ° C, gefolgt von 12 h, bei 32 ° C für 72 h), unterziehen bevor statt bei konstant 37 ° C für die Aufnahme unterzogen. 60 min Aufnahmen wurden stündlich genommen. Zwei von diesen Bedingungen sind in Abbildung 1 bquantifiziert.
Abbildung 2A zeigt eine schematische Darstellung der Einrichtung eines Systems zur perfundierten Gewebekultur. Diese besteht aus zwei Kanal Folien mit Schläuchen verbunden. Medien wird über die Zellen über eine Spritzenpumpe angetrieben. Die erste dieser Folien fungiert als eine durchlässige Blase Gassammelraum (Puffer Folie) und enthält keine Zellen, mit dem zweiten die Zellen auf dem Biolumineszenz aufgenommen. Ein Vertreter Videoaufnahme von diesem System ist in Abb. 2 bdargestellt. 15 min Aufnahmen wurden alle 15 Minuten. Hier werden Zellen gepflegt, standard Perfusion Bedingungen oder im Beisein von Kasein-Kinase-Inhibitor PF670462, das zuvor gezeigt worden ist, zu verlängern zirkadianen Periode und reduzieren die Amplitude der Uhr gen Ausdruck Rhythmen in kultivierten Säugerzellen14. Abbildung 2 (obere Abdeckung) zeigt die Auswirkung auf PER2::LUC Ausdruck gegen Zellen mit der gleichen Konzentration des Arzneimittels unter statischen Standardzelle Kulturbedingungen, dargestellt in Abbildung 2 (Bodenplatte) mit Quantifizierung der Zeitraum behandelt in Abb. 2Ddargestellt. Es ist daraus klar, dass die Behandlung mit PF670462 PER2::LUC Ausdruck unter beide Bedingungen beeinflusst. Während Zellen mit PF670462 behandelt unter perfundierten Bedingungen zeigen Periode Verlängerung von ca. 3 h (3 ±0.9 h), Zellen unter statischen Bedingungen mit dem Medikament behandelt zeigen jedoch deutlich mehr Zeit Verlängerung auf einen Zeitraum von &#62; 48 h. Dies kann durch eine gedämpfte Kosinus, fit sein, wie in Abschnitt 7 beschrieben (zusätzliche Summe der Quadrate F testen im Vergleich zu einer geraden Linie, p &#60; 0,0001). Interessanterweise ist das Ausmaß der Periode Verlängerung unter Perfusion viel näher an, die in Vivo14beobachtet.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56623/56623fig1v2.jpg"> 
Abbildung 1: Daten Beispiel. (A) Beispiel, das video-Schnappschuss der Biolumineszenz von verewigt PER2::LUC 6 x 96 auch im Inkubator Biolumineszenz Platten. Brunnen zu quantifizierenden wurden in der ergänzenden Video 1gelb hervorgehoben. (B) Quantifizierung der Biolumineszenz von A. Zwei Sätze von 3 Platten Zellen wurden mitgerissen mit Temperaturzyklen (12 h, bei 32 ° C; 12 h bei 37° C), die Anti-phasisch einander gegenüber Phasen der Proteinexpression PER2 produzieren waren (n = 6, bedeuten ± SEM). <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56623/56623fig1v2large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56623/56623fig2v2.jpg"> 
Abbildung 2: Perfusion mit CK1δ Inhibitoren. (A) schematische Darstellung der Perfusion System. (B) Beispiel video-Schnappschuss von einer Biolumineszenz, die Aufnahme von PER2::LUC unter Perfusion Zellen. Ein Probe-Bereich der Quantifizierung ist gelb markiert. (C) Quantifizierung der Biolumineszenz von PER2::LUC Fibroblasten unter Perfusion und unter statischen Bedingungen mit und ohne 3 µM CK1 Inhibitor PF670462 (n = 3, bedeuten ± SEM). Biolumineszenz ist auf minimalen und maximalen Werte normalisiert. (D) Analyse der Periode (Two-Way ANOVA, Holm-Sidak multiple Vergleiche-Test).<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56623/56623fig2v2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Komponente</td>
			<td>Mittlere Endkonzentration (μM)</td>
			<td>Lager (mg/mL)</td>
			<td>Für 400 mL NS21</td>
		</tr>
		<tr>
			<td>Bovine Albumin</td>
			<td>37</td>
			<td>-</td>
			<td>50 g</td>
		</tr>
		<tr>
			<td>Katalase</td>
			<td>0,01</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Glutathion</td>
			<td>3.2</td>
			<td>-</td>
			<td>20 mg</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>0,6</td>
			<td>10</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Superoxid-dismutase</td>
			<td>0.077</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>0,062</td>
			<td>-</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>T3 (Triiodo-L-Thyronine)</td>
			<td>0.0026</td>
			<td>2</td>
			<td>20 ΜL</td>
		</tr>
		<tr>
			<td>L-Carnitin</td>
			<td>12</td>
			<td>-</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td>Ethanolamin</td>
			<td>16</td>
			<td>Flüssigkeit (1 g/ml)</td>
			<td>20 ΜL</td>
		</tr>
		<tr>
			<td>(+) D-Galactose</td>
			<td>83</td>
			<td>-</td>
			<td>300 mg</td>
		</tr>
		<tr>
			<td>Putrescin</td>
			<td>183</td>
			<td>-</td>
			<td>322 mg</td>
		</tr>
		<tr>
			<td>Natriumselenit</td>
			<td>0.083</td>
			<td>1</td>
			<td>280 ΜL</td>
		</tr>
		<tr>
			<td>Corticosteron</td>
			<td>0.058</td>
			<td>2</td>
			<td>0,2 mL</td>
		</tr>
		<tr>
			<td>Linolsäure</td>
			<td>3.5</td>
			<td>100</td>
			<td>0,2 mL</td>
		</tr>
		<tr>
			<td>Linolensäure</td>
			<td>3.5</td>
			<td>100</td>
			<td>0,2 mL</td>
		</tr>
		<tr>
			<td>Liponsäure</td>
			<td>0,2</td>
			<td>4.7</td>
			<td>0,2 mL</td>
		</tr>
		<tr>
			<td>Progesteron</td>
			<td>0.02</td>
			<td>3.2</td>
			<td>0,04 mL</td>
		</tr>
		<tr>
			<td>Retinol Acetat</td>
			<td>0,2</td>
			<td>20</td>
			<td>0,1 mL</td>
		</tr>
		<tr>
			<td>Retinol, alle trans</td>
			<td>0,3</td>
			<td>10</td>
			<td>0,2 mL</td>
		</tr>
		<tr>
			<td>D, L-Alpha-Tocopherol</td>
			<td>2.3</td>
			<td>100</td>
			<td>0. 2 mL</td>
		</tr>
		<tr>
			<td>D, L-Alpha-Tocopherol Acetat</td>
			<td>2.1</td>
			<td>100</td>
			<td>0,2 mL</td>
		</tr>
	</tbody>
</table>
Tabelle 1: NS21 Vorbereitung.
Ergänzende Video 1: Beispielvideo der Biolumineszenz von well-Platten. Beispiel video-Schnappschuss der Biolumineszenz von verewigt PER2::LUC in 6 x 96-well-Platten im Inkubator Biolumineszenz. Brunnen zu quantifizierenden sind gelb markiert. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56623/Video_1A_Revision-1.avi">Bitte klicken Sie hier, um diese Datei herunterladen.</a>
Ergänzende Video 2: Beispiel-video-Schnappschuss von einer Biolumineszenz, die Aufnahme von PER2::LUC Zellen unter Perfusion. Ein Probe-Bereich der Quantifizierung ist gelb markiert. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56623/Video_2B_Revision-1.avi">Bitte klicken Sie hier, um diese Datei herunterladen.</a>

Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetisch codierte Luciferase ist eine beliebte nicht-invasive Reporter der Genexpression. Eine automatisierte längs Luciferase imaging-Gas - und Temperatur-optimierte Recorder (ALLIGATOR) können längs Aufnahme von Biolumineszenz Zellen unter den unterschiedlichsten Bedingungen. Hier zeigen wir, wie ALLIGATOR im Rahmen des circadianen Rhythmus Forschung verwendet werden können.

Flexible Messung der Biolumineszenz Reporter mit einer automatisierten längs Luciferase Imaging-Gas - und Temperatur-optimierte Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

flexible-measurement-bioluminescent-reporters-using-an-automated

Universidad San Sebastian

Research

JoVE Journal

Biology

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

7.8K Aufrufe.  MRC Laboratory of Molecular Biology. Genetisch codierte Luciferase ist eine beliebte nicht-invasive Reporter der Genexpression. Eine automatisierte längs Luciferase imaging-Gas - und Temperatur-optimierte Recorder (ALLIGATOR) können längs Aufnahme von Biolumineszenz Zellen unter den unterschiedlichsten Bedingungen. Hier zeigen wir, wie ALLIGATOR im Rahmen des circadianen Rhythmus Forschung verwendet werden können.

Serie: Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Gründung Intrakranielle Brain Tumor Xenografts mit anschließender Analyse des Tumorwachstums und Ansprechen auf die Therapie mit Biolumineszenz-Bildgebung

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Forschung

Neurowissenschaften

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Mit Luciferase to Image Bakterielle Infektionen bei Mäusen

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Immunologie und Infektion

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual-Substrat Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Medizin

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Studieren Membrane Biogenesis mit einem Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Biologie

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakterielle Imaging<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Ein Multiplex-Luciferase-basierte Screening-Plattform zur Abfrage Krebs-assoziierte Signaltransduktion in kultivierten Zellen

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Echtzeit-Biolumineszenz-Bildgebung der Notch-Signaldynamik während der murinen Neurogenese

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Entwicklungsbiologie

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

Überwachung Zell-autonome circadiane Uhr Rhythms der Genexpression mittels Luciferase Biolumineszenz Reporter

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

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

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

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 ...

Optimierung der menschlichen Darmenteroide für Studien zum zirkadianen Rhythmus

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Autor im Rampenlicht: In vitro Untersuchungen von zirkadianen Rhythmen in multizellulären Systemen

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Überwachung zirkadianer Oszillationen mit einem Biolumineszenz-Reporter in Organoiden

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...