This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

Wir stellen detaillierte Verfahren zur Abbildung lokalen Ca 2+ Ereignisse in menschlichen Neuroblastom-SH-SY5Y-Zellen. Diese Verfahren können zur Bild intrazellulärem Ca 2+ Signale in vielen Zelltypen 8-10 anpassen. 1. Herstellung der Zellen <ol><li> Kultur die Zellen gemäß den Anweisungen auf der Website ihren Lieferanten aufgeführt abgebildet werden. </li><li> Einige Tage vor der Bilderzeugung, Ernten der Zellen in einem Gewebekulturkolben unter Verwendung von 1 ml 0,25% Trypsin / EDTA (2-3 min) gezüchtet. Nach Ablösung, dasselbe Volumen der Kulturmedien, um das Trypsin zu inaktivieren. </li><li> Führen Sie eine Zellzahl mit einer Zählkammer und Samenzellen in Glasboden Bildgebung Schalen in einer Dichte von 3-7 × 10 4 Zellen pro Schale. Wählen Sie die Zellen, die für Imaging-Experimente, wenn sie erreichen 60% Konfluenz (2-3 Tage). </li></ol> 2. Herstellung von Lösungen und Reagenzien <ol><li> Bereiten Sie eine Ca 2+ -haltige HEPES gepuffertSalzlösung (Ca 2+ -HBSS) von (mM) aus: 135 NaCl, 5,4 KCl, 2 CaCl 2, 1 MgCl 2, 10 HEPES und 10 Glucose; pH = 7,4 bei Raumtemperatur (RT) mit NaOH. Bereiten Ca 2+ -HBSS ohne die Zugabe von Glucose und bei 4 ° C; Zugeben von Glukose zu der Lösung unmittelbar vor der Verwendung. </li><li> Löslich membrangängige Form des fluoreszierenden Ca 2+ Indikator Cal-520 (1 mM), CI-IP 3 (200 uM) und EGTA (100 mM) in Dimethylsulfoxid (DMSO), die 20% Pluronic F-127. Aliquot dieser Reagenzien in Eppendorf-Röhrchen und lagern, vor Feuchtigkeit und Licht geschützt, bei -20 ° C. </li></ol> 3. Loading-Zellen mit membrangängige Cal-520, ci-IP 3 und EGTA <ol><li> Bereiten Sie die &quot;Ladepuffer&quot; durch Verdünnen Stammlösungen von membrangängige Cal-520 und ci-IP 3 bis zu einer Endkonzentration von 5 uM und 1 &amp; mgr; M, in Ca 2+ -HBSS. </li><li> Saugen Sie culture Medien und spülen Zellen durch Ersatz von Medien mit Ca 2+ -HBSS dreimal. </li><li> Entfernen Ca 2+ -HBSS inkubieren Zellen in Ladepuffer für 60 min bei RT im Dunkeln. </li><li> Entfernen Ladepuffer und spülen Zellen durch Ersatz von Medien mit Ca 2+ -HBSS dreimal. <ol><li> Falls gewünscht, kann in diesem Schritt, Wägezellen mit EGTA / AM durch Verdünnen der Stammlösung auf eine Endkonzentration von 5 &amp; mgr; M Ca 2+ -HBSS und dann inkubieren Zellen 30-60 min bei RT im Dunkeln. Im Anschluss an diese Inkubation spülen Zellen dreimal mit Ca 2+ -HBSS bevor Sie mit Schritt 3.5. </li></ol></li><li> Zellen in Ca 2+ -HBSS Inkubation 30 min zur Entesterung von belasteten Reagenzien ermöglichen. </li><li> Unmittelbar vor der Abbildungs ​​Ersetzen Ca 2+ -HBSS mit &quot;frischen&quot; Ca 2+ -HBSS um jeden Farbstoff, der in der letzten Inkubation in das Bad ausgetreten sind, beseitigt werden. </li></ol> 4. Ca 2+ </sup&gt; Image Acquisition <ol><li> Setzen Sie einen kleinen Tropfen Immersionsöl auf die 100X APO TIRF Ziel (NA 1,49). </li><li> Montieren Sie die Bildform auf der Bühne des inversen Mikroskop und bringen die Zellen in den Fokus im Durchlicht. Es ist wichtig, um die Abbildungsschale, um eine Bewegung während der Aufnahme zu verhindern sichern. </li><li> Beleuchten Zellen mit einem 488 nm Laser, um Cal-520 erregt und sammeln emittierte Fluoreszenz (λ&gt; 510 nm) unter Verwendung eines Hochgeschwindigkeits EMCCD- Kamera. HINWEIS: Die Software, die das Mikroskop arbeitet erlaubt dem Benutzer, eine Fokussierlinse ermöglicht den Laserstrahl eingebracht werden, entweder an der äußersten Kante der Objektivrücköffnung (für TIRF Anregung) oder mehr in der Mitte (bei &quot;WF&quot; Anregung) zu übersetzen. </li><li> Mit der EMCCD-Software, reduzieren Sie die Bildfeld aus den vollen 512 x 512 Pixel, um Daten in der erforderlichen zeitlichen Auflösung zu sammeln, um lokalen Ca 2+ Ereignisse zu erfassen. HINWEIS: Zum Beispiel Zentrum QuadErwerb von 256 x 256 Pixel beschleunigt die Erfassungsrate bis 66 fps. Es ist auch möglich, die Bildrate des EMCCD Kamera zu 500 Bildern pro Sekunde weiter zu erhöhen mit einem Isolated Crop-Modus-Funktion. </li><li> Konfigurieren Sie die Software, um automatisch ein paar Sekunden der Grundaktivität, liefern einen UV-Blitz, um die Zellen und fährt mit der Aufnahme für ein weiteres 10 bis 30 sec. HINWEIS: Ein UV-Blitz wird mit einer durch eine UV-reflektierenden dichroitischen Spiegel in den rückseitigen Anschluss des Mikroskops eingeführt Xenon-Bogenlichtquelle geliefert. Die Dauer und Intensität der UV-Blitz wird empirisch eingestellt, um eine gewünschte Frequenz des lokalen Ca 2+ Ereignisse hervorrufen. </li><li> Speichern von Dateien als Bildstapel für die Offline-Analyse. </li></ol> 5. Automatische Ca 2+ Bildanalyse HINWEIS: Es ist möglich, zu sehen, zu verarbeiten und zu analysieren, die erfassten Daten mit vielen kommerziellen Softwarepaketen. Jedoch haben wir einen Algorithmus, um schnell zu automatisieren Identifikations eine entwickelted Analyse der lokalen Ca2 + Signale. Eine detaillierte Beschreibung der räumlichen und zeitlichen Filter in diesem Algorithmus verwendet wird, kann die Erzeugung von &amp; Dgr; F / F 0 und Identifizierung / Auswerteroutinen in 11 gefunden werden. Dieser Algorithmus wurde entwickelt, um auf dem Open-Source-Software-Plattform Python ausführen und erhalten werden, zusammen mit Beispiel experimentellen Daten und ausführliche Gebrauchsanweisung, per E-Mail an den entsprechenden Autor (ismith@uci.edu). <ol><li> Konvertieren von Dateien in das speziell geschriebenen Algorithmus, um die Mehrebenen-TIFF-Datei-Format importiert werden. </li><li> Suchen Sie den Ordner mit der Analyse-Algorithmus und doppelklicken Sie auf die run.exe. </li><li> Wählen Sie das TIFF-Datei analysiert werden. </li><li> Wählen Sie Analyseparameter im offenen Dialog. Siehe 2A für Standardwerte für die Probendaten. </li><li> Bestimmen Sie den Schwarzwert aus dem Bildstapel entweder durch Bewegen des Cursors auf einen Teil des Sichtfeldes, die n tut ausgeglichen werdenot enthalten eine Zelle (siehe Abbildung 2B) oder durch manuelle Eingabe eines Schwarzwert mit dem &quot;Schwarzpegel einstellen&quot; prompt. </li><li> Beobachten vier Fenster auf dem Bildschirm folgende Analyse durch die Software (2C-F). C ist ein monochromes Bild still Fluoreszenz von Zellen analysiert. Weiße Quadrate auf diesem Bild überlagert sind Veranstaltungsorte, wie die Schwerpunktpositionen der zweidimensionalen Gauß-Funktionen ausgestattet, um Ereignisse bestimmt. </li><li> Klicken Sie auf jedes Veranstaltungsortes, oder drücken Sie die Cursor-Tasten, um zwischen den Veranstaltungen. Daraufhin wird der Hintergrund subtrahiert, Gaussian geglättet Fluoreszenzverhältnis ändert (AF / F 0) auf jeder dieser Sites Update im Fenster D. HINWEIS: Rot markierte Ereignisse von &quot;Durchschlagen&quot; von Aktivität zu benachbarten Stellen lokalisiert entschlossen, von diesem bestimmten Ort stammt und nicht. Die untere blaue Linie zeigt an, wo das Fluoreszenzsignal an der ausgewählten Stelle über dem ErkennungsSchwelle, während die schwarze Linie kennzeichnet Events von diesem bestimmten Ort Ursprung (entspricht dem rot hervorgehoben Ereignisse). </li><li> Klicken Sie auf einen rot unterlegt Veranstaltung Fenster E, die die zeitliche Entwicklung der Veranstaltung zeigt, und Fenster F, zeigt das räumliche Profil der Veranstaltung, gemittelt über seine Zeit, natürlich zusammen mit der Raumprofil der angepassten Gaußfunktion aktualisieren. </li><li> Manuelles Überprüfen Sie die Ereignisse, die identifiziert wurden, so dass Artefakte von der Analyse abgelehnt. Löschen Sie solche Ereignisse durch Rechtsklick auf das rot markierte Veranstaltungen. HINWEIS: In unseren Händen, Ca 2+ Fluss durch einzelne IP 3 R-Kanäle erinnert Signale mit AF / F 0 etwa 0,11, so dass alle erkannt &quot;Ereignisse&quot; wesentlich kleiner als diese wahrscheinlich mit Fehlmeldungen rechnen. </li><li> Exportieren von Daten, indem Sie durch Ziehen um die Zelle von Interesse und Auswählen von »Save CellR&quot; Save to Excel &#39;oder Exportieren von Daten auf eine Basis pro Zelle17 ;. HINWEIS: Die Daten werden in den gleichen Ordner wie das Originalbild Stack gespeichert. </li></ol>

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	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1 (1), 11-21 (2000).</li><li>Hill-Eubanks, D. C., Werner, M. E., Heppner, T. J., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+smooth+muscle.">Calcium signaling in smooth muscle.</a> Cold Spring Harb Perspect Biol. 3 (9), a004549 (2011).</li><li>Hagenston, A. M., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+synapse-to-nucleus+communication.">Calcium signaling in synapse-to-nucleus communication.</a> Cold Spring Harb Perspect Biol. 3 (11), a004564 (2011).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elementary+and+global+aspects+of+calcium+signalling.">Elementary and global aspects of calcium signalling.</a> J Physiol. 499 (Pt 2), 291-306 (1997).</li><li>Lipp, P., Niggli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hierarchical+concept+of+cellular+and+subcellular+calcium+signalling).">A hierarchical concept of cellular and subcellular calcium signalling).</a> Prog Biophys Mol Biol. 65 (3), 265-296 (1996).</li><li>Parker, I., Yao, Y., Ilyin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+of+calcium+liberation+induced+in+Xenopus+oocytes+by+photoreleased+inositol+trisphosphate.">Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.</a> Biophys J. 70 (1), 222-237 (1996).</li><li>Minta, A., Kao, J. P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+cytosolic+calcium+based+on+rhodamine+and+fluorescein+chromophores.">Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.</a> J Biol Chem. 264 (14), 8171-8178 (1989).</li><li>Demuro, A., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+activity+and+localization+of+single+voltage-gated+Ca(2+)+channels+by+total+internal+reflection+fluorescence+microscopy.">Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy.</a> Biophys J. 86 (5), 3250-3259 (2004).</li><li>Smith, I. F., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+quantal+substructure+of+single+inositol+trisphosphate+receptor+channel+activity+during+calcium+puffs+in+intact+mammalian+cells.">Imaging the quantal substructure of single inositol trisphosphate receptor channel activity during calcium puffs in intact mammalian cells.</a> Proc Natl Acad Sci U S A. 106 (15), 6404-6409 (2009).</li><li>Reissner, K. 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=A+novel+postsynaptic+mechanism+for+heterosynaptic+sharing+of+short-term+plasticity.">A novel postsynaptic mechanism for heterosynaptic sharing of short-term plasticity.</a> J Neurosci. 30 (26), 8797-8806 (2010).</li><li>Ellefson, K. S., Parker, B., Smith, I., F, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+algorithm+for+automated+detection,+localization+and+measurement+of+local+calcium+signals+from+camera-based+imaging.">An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.</a> Cell Calcium. , (2014).</li><li>Smith, I. F., Wiltgen, S. 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The authors declare that they have no competing financial interests.

Wir beschreiben hier Protokolle zur Abbildung lokalen Ca 2+ Ereignisse in kultivierte Säugerzellen unter Verwendung von fluoreszierenden Ca 2+ Indikatoren. Darüber hinaus beschreiben wir einen Algorithmus mit einer intuitiven Benutzeroberfläche, die Identifikation und Analyse von erfassten Daten automatisiert. Das hier beschriebene Verfahren nutzen die fluoreszierenden Ca 2+ Anzeige Cal-520, aber viele andere Ca 2+ empfindlichen Farbstoffe wie Fluo-3, Fluo-4, Fluo-8 und Ore...

Calciumionen (Ca 2+) ubiquitär regulieren ein breites Spektrum von biologischen Prozessen, einschließlich Genexpression, Sekretion und dauerhafte Veränderungen in der synaptischen Plastizität 1. Ein Weg, durch welche Ca 2+ in einer vielfältigen Art und Weise wirken, ist durch die unterschiedliche räumliche und zeitliche Muster von Ca 2+ signalisiert eine Zelle erzeugen kann. Zum Beispiel globale Erhebungen in zytosolischen [Ca 2+] Trigger Kontraktion in glatten Muskulatur 2 während kleinere, lokale vorübergehende Erhöhung (lokalen Ca 2+ Mikrodomänen) zu stimulieren Genexpression essentiell für Lernen und Gedächtnis 3. Freien cytosolischen [Ca 2+] liegt bei ~ 100 nM in Ruhe gehalten wird, kann jedoch schnell zu mehreren Mikro molaren nach der Einstrom von Ca 2+ in das Cytosol steigen durch Ca 2+ durchlässige Ionen-Kanäle in der Plasmamembran und befindet die Freisetzung von Ca2 + aus intrazellulären stores. Unser Labor konzentriert sich auf den Inositol-1,4,5-triphosphat-Rezeptor (IP 3 R), das eine Ca 2+ -Freisetzung Kanal im endoplasmatischen Retikulum (ER) membran bildet. Bei der Bindung von sowohl IP 3 und Ca 2+ auf die cytosolische Aktivierung Stellen des Rezeptors, öffnet das IP 3 R-Kanal zu befreien Ca 2+ in das ER-Lumen sequestriert. Die Freisetzung von Ca 2+ räumlich auf eine kleine Gruppe von IP 3 Rs begrenzt bleiben, um eine lokale Mikrodomäne cytosolischen Ca 2+ zu erzeugen (Ca 2+ puff 4) oder, abhängig von der Nähe der benachbarten Clustern von IP 3 R, kann verbreiten sich eine Zelle durch die Rekrutierung von mehreren puff Websites durch einen Prozess der Ca 2+ -induzierte Ca 2+ -Freisetzung (CICR) 5,6. Die Einführung von Kleinmolekül-Fluoreszenz-Ca 2+ Indikatorfarbstoffe von Roger Tsien entwickelte 7, verbunden mit modernster Mikroskopie imaging Techniken hat erheblich erleichtert unser Verständnis von Ca 2+ Signalisierung. Jüngste Fortschritte in der Kameras für die Mikroskopie verwendet nun zur Abbildung vorübergehenden lokalen Ca 2+ Ereignisse wie Windbeutel mit beispielloser räumlicher und zeitlicher Auflösung zu ermöglichen. Derzeit verfügbare EMCCD-Kameras ermöglichen Bildgebung mit 128 x 128 Pixeln bei&gt; 500 Bilder sec -1 (fps) und die neue Generation der komplementären Metall-Oxid-Halbleiter (CMOS) Kameras bieten eine höhere Pixel-Auflösung und sogar höhere Geschwindigkeit auf Kosten etwas höher Geräuschpegel. In Verbindung mit der Totalreflexion (TIRF) Mikroskopie ist es nun möglich, einzelne Bild Ca 2+ Kanalereignisse 8,9. Dieser Ansatz ermöglicht die Abbildung von Hunderten von Kanälen / Ereignisse gleichzeitig, während die Erzeugung von großen Datenmengen (ca. 1 GB pro Minute), die manuelle Bearbeitung, visuelle Identifizierung und Analyse machen unpraktikabel und setzen Sie eine Last auf die Entwicklung von automatisierten Algorithmen. 2+ Signale in intakten Säugerzellen unter Verwendung von fluoreszierenden Ca 2+ Indikatoren präsentieren wir. Wir zeigen ferner einen Algorithmus in der Open-Source-Umfeld Python, die Identifikation und Analyse von lokalen Ca 2+ Ereignisse sowohl von TIRF und konventionellen Weitfeld-Auflicht-Fluoreszenz (WF) Mikroskopie abgebildet automatisiert entwickelt. Obwohl wir beschreiben diese Ansätze im Zusammenhang mit der IP 3 -generated Ca 2+ Signalen leicht zu einer lokalen Veränderung in cytosolische [Ca 2+] aus einer Vielzahl von Ca 2+ ausgeh -durchlässigen Ionenkanäle entweder in der Oberfläche angeordnet zu untersuchen sind Membran oder intrazellulären Organellen 8-10.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:813px;">Name</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalogue No.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Neuroblastoma SH-SY5Y cells</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cal-520/AM</td>
			<td>AAT Bioquest Inc.</td>
			<td>21130</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl)</td>
			<td>SiChem</td>
			<td>cag-iso-2-145-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO/20% pluronic F127</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA/AM</td>
			<td>Invitrogen</td>
			<td>E-1219</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom imaging dishes</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
		</tr>
	</tbody>
</table>

imaging local ca2+ signals in cultured mammalian cells

Zytosolischen Ca 2+ -Ionen regulieren zahlreiche Aspekte der Zellaktivität in fast allen Zelltypen, Kontrollprozesse, wie vielfältig wie Gen-Transkription, elektrischen Erregbarkeit und Zellproliferation. Die Vielfalt und Besonderheit der Ca 2+ Signal leitet sich von Mechanismen, durch die Ca 2+ Signale erzeugt werden, um über verschiedene Zeit- und Raumskalen wirken, von der Zelle weiten Schwingungen und Wellen über die Perioden von Minuten, um lokale transiente Ca 2+ Mikrodomänen auftreten (Ca 2+ Hauche) anhalt Millisekunden. Jüngste Fortschritte in der Elektronen multipliziert CCD (EMCCD) Kameras jetzt Bildgebung von lokalen Ca 2+ Signale mit einem 128 x 128 Pixel Ortsauflösung ermöglichen mit Raten von&gt; 500 Bilder sec -1 (fps). Dieser Ansatz ist sehr parallel und ermöglicht die gleichzeitige Überwachung von Hunderten von Kanälen oder Blätterteig Stellen in einem einzigen Experiment. Doch die große Datenmengen generiert (ca. 1 GB per min) machen visuelle Identifizierung und Analyse der lokalen Ca 2+ Ereignisse nicht praktikabel. Hier beschreiben und zeigen die Verfahren für die Erfassung, Erkennung und Analyse der lokalen IP 3 vermittelte Ca 2+ Signale in intakten Säugerzellen mit Ca 2+ Indikatoren sowohl mit Weitfeld-Auflicht-Fluoreszenz (WF) und Totalreflexion geladen Fluoreszenz (TIRF) Mikroskopie. Darüber hinaus beschreiben wir einen Algorithmus in der Open-Source-Software-Umgebung Python, die die Identifizierung und Analyse dieser lokalen Ca 2+ Signale automatisiert entwickelt. Der Algorithmus lokalisiert Websites von Ca 2+ Freisetzung mit Subpixel-Auflösung; ermöglicht dem Benutzer Überprüfung der Daten; und gibt Zeitsequenzen der Fluoreszenzverhältnissignale mit Amplituden und kinetische Daten in einer Excel-Tabelle kompatibel.

1A zeigt eine WF Bild ruhender Cal-520-Fluoreszenz in menschlichen Neuroblastom-SH-SY5Y-Zellen ebenfalls mit ci-IP 3 geladen. Exposition dieser Zellen in einen 100 msec UV-Blitz, um foto Freisetzung i-IP 3 ausgelöste Ca 2+ Transienten Quasten an diskreten Stellen (durch die weißen Kreise in 1A angegeben). Fluoreszenz Spuren gemessen an diesen Stellen zeigten eine schnelle Anstiegsphase aufgrund transienter Öffnungen der IP 3 R, gefolgt von ...

Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Lokale Ca<sup&gt; 2+</sup&gt; Signale in kultivierten Säugetierzellen

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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Imaging Local Ca2+ Signals in Cultured Mammalian Cells

10.7K Aufrufe.  University of California, Irvine. Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Serie: Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Langfristige Imaging Säugetierzellen mit Wide-Field-Mikroskopie

Forschung

Biologie

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Live-Imaging von Dense-Core-Vesikel in Primary hippokampalen Neuronen

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques for Imaging Ca 2 + Signaling in menschlichen Spermien

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

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

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

MISSION esiRNA für RNAi-Screening in Säugerzellen

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

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse des Zellzyklus Position in Säugerzellen

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Automatisierte Analyse von dynamischen Ca<sup&gt; 2 +</sup&gt; Signale in Bildsequenzen

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA Screening zu Ubiquitin und Ubiquitin-ähnlichen System Regler der biologischen Stoffwechselwege in kultivierten Säugerzellen identifizieren

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Heme Synthesis Levels in Mammalian Cells

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

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

Die Messung der Hämsynthese Levels in Säugerzellen

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

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.