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.

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	<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. 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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. 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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 border="0" cellpadding="0" cellspacing="0" width="647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:145px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hyclone FetalClone III Serum</td>
			<td style="width:145px;">GE Healthcare</td>
			<td>SH30109.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:145px;">Thermofisher</td>
			<td>21103049</td>
			<td>basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:145px;">Sigma</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Catalase</td>
			<td style="width:145px;">Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutathione</td>
			<td style="width:145px;">Sigma</td>
			<td>G6013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin</td>
			<td style="width:145px;">Sigma</td>
			<td>I1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superoxide Dismutase</td>
			<td style="width:145px;">Sigma</td>
			<td>S5395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Holo-transferrin</td>
			<td style="width:145px;">Calbiochem</td>
			<td>616424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T3 (triiodo-L-thyronine</td>
			<td style="width:145px;">Sigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Carnitine</td>
			<td style="width:145px;">Sigma</td>
			<td>C7518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanolamine</td>
			<td style="width:145px;">Sigma</td>
			<td>E9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D (+)-Galactose</td>
			<td style="width:145px;">Sigma</td>
			<td>G0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Putrescine</td>
			<td style="width:145px;">Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Selenite</td>
			<td style="width:145px;">Sigma</td>
			<td>S9133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Corticosterone</td>
			<td style="width:145px;">Sigma</td>
			<td>C2505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linoleic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>L1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linolenic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>L2376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipoic Acid</td>
			<td style="width:145px;">Sigma</td>
			<td>T1395</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Progesterone</td>
			<td style="width:145px;">Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retinol Acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>R7882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Retinol, all trans</td>
			<td style="width:145px;">Sigma</td>
			<td>95144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D,L-alpha-Tocopherol</td>
			<td style="width:145px;">Sigma</td>
			<td>95240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D,L-alpha-Tocopherol acetate</td>
			<td style="width:145px;">Sigma</td>
			<td>T3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Bicarbonate Solution</td>
			<td style="width:145px;">Sigma</td>
			<td>S8761-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GlutaMAX (100x)</td>
			<td style="width:145px;">Gibco</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:145px;">Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galaxy 170R incubator&#160;</td>
			<td style="width:145px;">Eppendorf</td>
			<td style="width:119px;">CO17301001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin</td>
			<td style="width:145px;">Biosynth</td>
			<td style="width:119px;">L-8220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D -(+)-Glucose solution</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">G8644-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">D5030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MOPS</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">PHG0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mm I.D. silicone tubing&#160;</td>
			<td style="width:145px;">GE Healthcare</td>
			<td>19-4692-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Elbow luer connector</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male luer fittings</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Female luer fittings</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">10825</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#181;-slide luer I 0.6</td>
			<td style="width:145px;">Ibidi</td>
			<td style="width:119px;">80196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD plastipak 20ml syringe</td>
			<td style="width:145px;">Becton Dickinson</td>
			<td style="width:119px;">300613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1mm I.D. ETFE tubing</td>
			<td style="width:145px;">GE Healthcare</td>
			<td style="width:119px;">18-1142-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PF670462</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">SML0795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 Supplement (50x)</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:119px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iXon Ultra EMCCD camera</td>
			<td style="width:145px;">Andor</td>
			<td style="width:119px;">iXon 888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fiji</td>
			<td style="width:145px;">ImageJ</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prism 7.0</td>
			<td style="width:145px;">Graphpad Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan blue</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deltaphase Isothermal Pad</td>
			<td style="width:145px;">Braintree Scientific</td>
			<td style="width:119px;">39DP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heated neutral density filter&#160;</td>
			<td style="width:145px;">Cairn Research</td>
			<td style="width:119px;">Custom item</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmomat 030</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">Discontinued</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">300 mOsmol/kg calibration standard</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">30.9.0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Measuring vessel</td>
			<td style="width:145px;">Gonotech</td>
			<td style="width:119px;">30.9.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Focusing cylinder</td>
			<td style="width:145px;">Cairn Research</td>
			<td style="width:119px;">Custom item</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NE-1600 programmable syringe pump</td>
			<td style="width:145px;">Pump Systems inc.</td>
			<td style="width:119px;">NE-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor Solis Software</td>
			<td style="width:145px;">Andor</td>
			<td style="width:119px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

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

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

In vivo Biolumineszenz Imaging von Brusttumoren mit IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Forschung

Biologie

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Entwicklung von automatisierten Imaging und Analyse für Zebrafisch chemische Bildschirme.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

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 ...

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools5,6. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS)7. Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices8. To date, leaf shape programs (e.g. SHAPE9, LAMINA10, LeafAnalyzer11, LEAFPROCESSOR12) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size13. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Demonstration of key methods for high throughput leaf measurements. These methods can be used to accelerate leaf phenotyping when studying many plant mutants or otherwise screening plants by leaf phenotype.

LeafJ: Ein ImageJ Plugin für Semi-automatisierte Blattform Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review1 and recent examples2-4). Leaves are the ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Semi-Automated Imaging von Gewebespezifische Fluoreszenz-in Zebrafischembryonen

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Die Messung der extrazellulären Ionenflüsse Verwendung der ionenselektiven selbst verweisMikroElektroden-Technik

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Vorbereitung und Longitudinal Imaging der Herzfunktion in vivo unter Verwendung optischer Kohärenzmikroskopie (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

Eine effiziente und flexible Zellaggregationsmethode für 3D-Sphäroid-Produktion

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Messung des Zusammenflusses von iPSCs mit einem automatisierten Bildgebungssystem.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...