Diese Arbeit wurde von der National Science Foundation (Grant No. DBI 0519984) und die National Institutes of Health (Grant No. 5R01GM078279) unterstützt ..

1) Herstellung des pflanzlichen Materials <ol><li> Protoplasten können aus vielen verschiedenen Pflanzenarten und Geweben, sofern die richtige Mischung aus Zellwand verdauenden Enzyme 1 wird verwendet, abgeleitet werden. Bevor eine groß angelegte Experiment durchgeführt wird, ist eine kleine Verdauung des Materials empfohlen, um die Protoplastierung Effizienz des Gewebes, Enzyme, etc. zu bewerten und die Prozent-positiven Zellen für Zellsortierung zu schätzen. Hier Protoplasten aus den Wurzeln von Arabidopsis thaliana Keimlinge, die Zelltyp-spezifisch ausdrücken grün fluoreszierende Protein (GFP) eingesetzt werden abgeleitet. Die Endodermis und ruhenden Zentrum von P SCR markiert:: GFP und den ruhenden Mittelpunkt von P WOX5:: GFP 2,3 (Abbildung 1). </li><li> Sämlinge sind hydroponically in phytatrays gewachsen (Sigma; Abbildung 2a) auf eine Nylon-Filter (250 &amp;mgr; m mesh; NITEX), die die Wurzeln wachsen durch in das Wachstumsmedium (0,22% w / v Murashige und Skoog Basal Medium [Sigma], 1% lässt w / v Saccharose, 0,05% w / v MES [2 - (N-Morpholino) ethansulfonsäure], pH 5,7 mit KOH). Alternativ können die Pflanzen auf einer Nylon-Filter (100 um Maschenweite) in vertikal angeordneten 1% Agar-Platten (Abb. 2b) angebaut werden. </li><li> Die Verwendung der oben genannten Filter hilft nicht nur bei der Ernte der Wurzeln, es erleichtert auch eine zusätzliche Behandlung der Sämlinge, wenn gewünscht. Die Filter ermöglichen die Sämlinge en masse, neue phytatrays oder Agar-Platten mit einem Katalysator von Interesse ergänzt übertragen werden. Zum Beispiel die phytatray eingerichtet hat Zelltyp-spezifische Analyse der transkriptionellen Antwort auf Stickstoff-Behandlung in Arabidopsis-Keimlinge 4 verwendet. </li><li> Stellen Sie sicher, dass Ihre mikroskopisch Fluoreszenzmarker korrekt ausgedrückt ist (insbesondere bei Verwendung der Behandlungsmöglichkeit, da die Zelltyp-spezifische Marker sich durch die Behandlung beeinflusst sein könnten). In diesem Fall sind Sämlinge unter einem Fluoreszenzmikroskop (; Abbildung 1 Nikon) inspiziert. Beachten Sie, dass Zelltyp-spezifischen fluoreszierenden Marker Linien zunächst sollte unter einem konfokalen Mikroskop genau, welche Zelltypen sind markiert ermitteln und festzustellen, Variabilität im Ausdruck gekennzeichnet werden. </li></ol> 2) Herstellung der Protoplastierung Lösung <ol><li> Dissolve 1,25% w / v Cellulase (Yakult), 0,3% w / v Macerozyme (Yakult), 0,4 M D-Mannitol, 20 mM MES, 20 mM KCl (aus einer 1 M Stammlösung) in VE-Wasser und den pH-Wert auf 5,7 mit 1 M Tris / HCl pH 7,5. Diese Lösung wird leicht getrübt werden. </li><li> Erhitzen Sie die Lösung auf 55 ° C für 10 Minuten (die Lösung wird wiederum klar) und abkühlen lassen auf Raumtemperatur, bevor die Zugabe von 0,1% w / v BSA (Rinderserumalbumin), 10 mM CaCl 2, und 5 mM β-Mercaptoethanol . </li></ol> 3) Ernte und Protoplastierung des Pflanzenmaterials <ol><li> Die Wurzeln sind durch Kratzen sie aus dem Nylonnetz mit einem Skalpell geerntet und hinterlegt in einen Kolben mit dem Protoplastierung Lösung. Im Allgemeinen ist 10 ml Protoplastierung Lösung pro 1.500 Keimling Wurzeln verwendet. </li><li> Schütteln Sie die Kolben vorsichtig (75 rpm) bei Raumtemperatur für eine Stunde. Eine längere Inkubationszeit kann sich die Protoplasten Ertrag, sondern auch um die Wirkung von Protoplastierung sich auf die Genexpression hinzuzufügen. </li><li> Filtern Sie die Protoplasten-Lösung mit einem 40 um Zellsieb (BD Falcon) und teilen sich die Lösung über konische 15 ml Röhrchen (BD Falcon). </li><li> Drehen Sie das Röhrchen in einem Ausschwingrotor Zentrifuge für 10 Minuten bei 500 G. Beachten Sie, dass dieser Zentrifugation Geschwindigkeit hängt von der Art des Protoplasten verwendet, ihre Zerbrechlichkeit und die Höhe der Zelltrümmer während enzymatische Behandlung produziert wird. </li><li> Entfernen Sie die meisten der Überstand resuspendieren die Protoplasten in der verbleibenden Lösung und untersuchen diese mikroskopisch (Abbildung 3). </li><li> Nutzen Sie die Hämazytometer, um die Anzahl und Dichte der Protoplasten zu schätzen. Die Zelldichte bestimmt Probeninjektion Geschwindigkeit, die Ereignisse pro Sekunde und damit die gesamte Zeit benötigt, um die Zellen bei der FACS-Sortierung (siehe auch Abschnitt 4.3). </li><li> 3,7) Entweder gehen Sie direkt zu FACS oder waschen und resuspendieren die Protoplasten in einer Inkubationslösung, wie W5 (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM MES, pH-Wert einstellen auf 5,7 mit KOH) oder Protoplastierung Lösung ohne zugesetzten Enzyme. Besonders, wenn man transkriptionelle Veränderungen ist es von Bedeutung, um die Exposition der Proben, die Bedingungen, die die Genexpression beeinflussen können, wie das Ändern der Puffer der Protoplasten in. Es wird daher empfohlen, den Protoplasten in Protoplastierung Lösung zu halten und weiter gehalten werden minimiert zu FACS so schnell wie möglich. </li></ol> 4) Fluorescence Activated Cell Sorting von Protoplasten <ol><li> Schalten Sie und bereiten die Zell-Sorter. Hier wird ein FACSAria (BD) verwendet. </li><li> Einrichten einer Strömung mit einem 100 &amp; mu, m Düse und einem 20 psi Mantel Druck. </li><li> Die Zelldichte und Probenaufgabe Geschwindigkeit an die jeweilige Experiment, ob eine bestmögliche Ausbeute oder schnellste erreichbare Geschwindigkeit gewünscht wird, basiert eingestellt werden. Wir haben erfolgreich mit Dichten bis zu 10.000.000 Zellen / ml sortiert. </li><li> Verwenden Sie die Probe Agitation Option auf der FACS zur Sedimentation der Protoplasten zu verhindern. Wenn das Verstopfen der FACS ist ein Problem, gibt es drei mögliche Schritte zur Fehlerbehebung: 1. Führen Sie eine Probe-line Rückspülung. 2. Verdünnen Sie Ihre Protoplastensuspension, um die Dichte zu reduzieren. 3. Clean up the Protoplasten Lösung durch Wiederholung der Filtration (3,3) nach Zentrifugation und Resuspension. </li><li> Bereiten Sie das Gerät auf forward scatter (FSC), Side Scatter (SSC) und Emission bei 530/30 nm für GFP und 610/20 nm für Rot Spektrum Autofluoreszenz (RSA) nach Anregung durch einen 488 nm Laser zu messen. Dies sind im Wesentlichen die einzigen Parameter verwendet, um GFP-positive Protoplasten isolieren. Hier wurden die Spannung Einstellungen wie folgt: FSC - 60V, SSC 250V, 350V und GFP RSA 335V. Beachten Sie, dass die optimale Spannung Einstellungen werden bei jedem anders FACS und sogar müssen während der gesamten Lebensdauer des Zellsortierers eingestellt werden. </li><li> Starten Sie durch die Einrichtung einer Dotplot für Vorwärtsstreuung gegenüber side scatter. Übernehmen Sie die Spannung so, dass die gemessenen Ereignisse in die Handlung zentriert sind. </li><li> Anschließend erstellen Sie eine Dot-Plot von grünen und roten Fluoreszenz-Signale. Übernehmen Sie die Spannung so, dass die gemessenen Ereignisse eine zentrierte diagonal Bevölkerung in die Handlung ergeben, wenn man eine Wildtyp-(non-GFP) Protoplastensuspension. Ein Protoplastensuspension aus einer GFP-Marker Linie abgeleitet wird einen klaren Bevölkerung des grün fluoreszierenden Ereignisse nie in Wildtyp-Proben zu sehen. </li><li> Set Entschädigung Einschränkungen für spektrale Überlappung zwischen GFP und RSA einstellen. Angemessene Entschädigung Einschränkung Einstellungen werden für eine bessere Trennung der GFP-positive Protoplasten aus dem nicht-GFP Protoplasten und Schutt zu ermöglichen. Die Einschränkungen werden dabei wie folgt: RSA, minus 17,91% GFP. </li><li> Richten Sie ein Tor zur GFP-positive Ereignisse, eine negative Kontrolle von Nicht-GFP Protoplasten verwendet werden, um bei der Festlegung der Grenzen Tor (Abbildung 4) Hilfe soll zu identifizieren. </li><li> Implementieren Sie eine Vorwärtsstreuung Cutoff, um kleine Fremdkörper aus der Analyse zu verlassen. Visualisieren Sie die GFP-positive Ereignisse in der FSC vs SSC Plot um die Platzierung der Cutoff. Hier wurde der Cutoff bei 5.000 gesetzt. Beachten Sie, dass die FACS wird Schmutz als eine Art Veranstaltungen und eine Probe mit einem hohen Grad von Schmutz rechnen können eine unterschiedliche Prozent GFP positive Ereignisse als erwartet. Dies ist nicht unbedingt ein Problem. Allerdings, je mehr Schmutz in die Probe, desto länger die Art stattfinden wird. </li><li> Je nach Experiment und die Fülle der Zelltyp zu analysieren, stellen Sie die FACS Präzisions-Modus entweder für eine optimale Ausbeute oder die optimale Reinheit der sortierten Zellen. </li><li> Für die RNA-Extraktion, bereiten Sammelröhrchen (1,5 ml Mikrozentrifugenröhrchen) mit der entsprechenden Menge an RNA-Extraktionspuffer. Mit diesem Setup werden 20.000 Sortierung Ereignisse ergeben ein Gesamtvolumen von rund 100 ul, die in auf 350 ul Extraktionspuffer (RNeasy Micro Kit, QIAGEN) sortiert wurden. Mix Proben nach Abschluss der Art, wie Zellsuspension Pool an der Spitze. </li><li> Shop-Proben oder gehen Sie direkt zur Extraktion RNA. Erfolgreiche Microarray-Analysen wurden mit der RNA aus so wenig wie 500 Sortieren Ereignisse extrahiert wurden vorgeformt. Hier haben wir eine RNeasy Micro Extraction Kit (QIAGEN), der WT-Ovation Pico RNA Amplification System und FL-Ovation cDNA Biotin-Modul V2 (NuGEN). </li></ol> Repräsentative Ergebnisse Ein phytatray von etwa 1.500 Ein-Wochen alten P SCR:: GFP Keimlinge ergab rund 60.000 Protoplasten (wie von Hämazytometer gemessen). 2,6% von 65.000 FACS-verarbeiteten Ereignisse wurden als GFP-positiv definiert und sortiert wurden (Abbildung 4b). Acht Platten von etwa 1.500 Vier-Tage-alten P WOX5:: GFP Sämlinge jeder (12.000 insgesamt) ergab ca. 30.000.000 Protoplasten (wie von Hämazytometer gemessen). 0,063% von 16.000.000 FACS-verarbeiteten Ereignisse wurden als GFP-positive definiert und sortiert werden (Abbildung 4c). 10.000 sortiert Ereignisse sind in der Regel für die RNA-Extraktion verwendet und kann von 20 bis 140 ng Gesamt-RNA (Abbildung 5) ergeben. <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Zelltyp-spezifischen GFP Markierungslinien in der Arabidopsis root. Fluoreszenzmikroskopie Bilder wurden mit Differential-Interferenz-Kontrast (DIC) und GFP-Filter auf eine Eclipse 90i-Mikroskop (Nikon), die auf Metamorph-Software (Molecular Devices) übernommen. Die DIC-und GFP-Bilder wurden zur Visualisierung überlagert. Die beiden Markierungslinien in dieser visuellen Experiment verwendet werden gezeigt;a) P SCR:: GFP und b) P WOX5:: GFP. <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Das Pflanzenwachstum Bedingungen. Sämlinge wurden in ein Umwelt-Controller hydroponically in phytatrays (a) oder vertikal positioniert Agarplatten (b) gewachsen. <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Pflanzenprotoplasten, die GFP. Fluoreszenzmikroskopie Bilder wurden mit Differential-Interferenz-Kontrast (DIC) und GFP-Filter auf eine Eclipse 90i-Mikroskop (Nikon), die auf Metamorph-Software (Molecular Devices) übernommen. Die DIC-und GFP-Bilder wurden zur Visualisierung überlagert. Die Pfeile zeigen eine Burst-Zelle, Zelltrümmer und einem GFP-positive Protoplasten. Der Abstand zwischen zwei weißen Linien ist 50 pm. <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Fluorescence Activated Cell Sorting von GFP-positive Protoplasten Protoplasten aus dem Wildtyp (a) P SCR abgeleitet.:: GFP (b) oder P WOX5:: GFP (c) Markierungslinien wurden analysiert und mit einem FACSAria (BD) sortiert mit Toren definiert auf einem Dotplot von grün (530/30 nm; x-Achse) gegen Rot (610/20 nm; y-Achse) Fluoreszenz. 100.000 Veranstaltungen sind in jeder Parzelle vorgestellt. Die Ereignisse, die in den GFP Weiche sind grün markiert. <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Vertreter RNA Extraktion von 10.000 sortierten Zellen. Die Zellen wurden direkt in RNA-Extraktionspuffer (QIAGEN) sortiert wurde die RNA gereinigt und geprüft für Konzentration, Reinheit und Integrität auf einem 2100 Bioanalyzer (Agilent). Drei Wiederholungen für beide Marker-Linien angezeigt werden.

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

Protoplasten können im Prinzip aus einer Vielzahl von pflanzlichen Geweben abgeleitet werden, die Optimierung der günstigen Bedingungen ermöglicht eine wesentlich bessere RNA Qualität und Quantität. Sowohl die Protoplastierung Lösung und die Wahl Inkubationspuffer beeinflussen diesen Aspekt. Viele verschiedene fluoreszierende Proteine ​​verwendet werden können, je nach den Fähigkeiten der FACS verwendet werden, zB GFP, RFP, YFP, CFP oder ihren vielen Varianten und Deriva...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence activated cell sorting of plant protoplasts

Hochauflösende, Zelltyp-spezifische Analyse der Genexpression stark erhöht das Verständnis von Entwicklungs-Regelung und Antworten auf Reize aus der Umwelt in allen vielzelligen Organismus. In-situ-Hybridisierung und Reporter-Gen-Visualisierung kann in begrenztem Umfang zu diesem Zweck verwendet werden, aber für hochauflösende quantitative RT-PCR oder High-Throughput-Transkriptom-weite Analyse der Isolierung von RNA aus bestimmten Zelltypen ist Voraussetzung. Cellular Dissoziation von Gewebe exprimiert ein fluoreszierendes Protein-Marker in einem bestimmten Zelltyp und anschließende Fluorescence Activated Cell Sorting (FACS) macht es möglich, ausreichende Mengen an Material für die RNA-Extraktion, cDNA-Synthese /-Amplifikation und Microarray-Analyse zu sammeln. Ein umfangreiches Set von Zelltyp-spezifischen fluoreszierenden Reporter-Linien ist für die Pflanze verfügbar Forschungsgemeinschaft. In diesem Fall sind zwei Markierungslinien der Arabidopsis thaliana Wurzel verwendet: P SCR:: GFP (Endodermis und ruhenden Mitte) und P WOX5:: GFP (Ruhe-Center). Große Zahlen (in Tausend) der Sämlinge sind hydroponically oder auf Agarplatten gewachsen und geerntet, um genügend root Material für weitere Analysen zu erhalten. Cellular Dissoziation von Pflanzenmaterial wird durch enzymatische Verdauung der Zellwand erreicht. Dieses Verfahren nutzt hohe Osmolarität induzierte Plasmolyse und kommerziell erhältliche Cellulasen, um Pektinasen und Hemicellulasen Protoplasten in Lösung freigeben. FACS von GFP-positiven Zellen nutzt die Visualisierung der grünen gegen die roten Emissionsspektren von Protoplasten von einem 488 nm Laser angeregt. GFP-positive Protoplasten können durch ihre erhöhte Verhältnis von Grün auf Rot-Emission unterschieden werden. Protoplasten werden in der Regel direkt in RNA-Extraktionspuffer sortiert und für die weitere Verarbeitung zu einem späteren Zeitpunkt. Diese Technik wird enthüllt werden einfach und praktikabel ist. Darüber hinaus wird gezeigt, dass sie ohne Schwierigkeiten eine ausreichende Anzahl von Zellen für die Transkriptom-Analyse zu isolieren können verwendet werden, auch bei sehr knappen Zelltypen (zB Ruhe-Zentrum-Zellen). Schließlich ist ein Wachstumsmarkt Setup für Arabidopsis-Keimlinge zeigten, dass ermöglicht eine unkomplizierte Behandlung der Pflanzen vor Zellsortierung (zB für die Zelltyp-spezifische Analyse der biotischen und abiotischen Stress-Reaktionen). Mögliche zusätzliche Einsatzmöglichkeiten für FACS von pflanzlichen Protoplasten werden diskutiert.

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

Ein Verfahren zur Isolierung von spezifischen Zelltypen aus pflanzlichem Material nachgewiesen ist. Diese Technik bedient sich transgene Marker exprimieren fluoreszierenden Proteinen in bestimmten Zelltypen, Zell-Dissoziation und Fluorescence Activated Cell Sorting. Zusätzlich wird ein Wachstum Setup hier erleichtert Behandlung von etablierten<em&gt; Arabidopsis thaliana</em&gt; Setzlinge vor cell sorting.

Fluorescence Activated Cell Sorting von Pflanzenprotoplasten

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

fluorescence-activated-cell-sorting-of-plant-protoplasts

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24.7K Aufrufe.  New York University. Ein Verfahren zur Isolierung von spezifischen Zelltypen aus pflanzlichem Material nachgewiesen ist. Diese Technik bedient sich transgene Marker exprimieren fluoreszierenden Proteinen in bestimmten Zelltypen, Zell-Dissoziation und Fluorescence Activated Cell Sorting. Zusätzlich wird ein Wachstum Setup hier erleichtert Behandlung von etablierten Arabidopsis thaliana Setzlinge vor cell sorting.

Serie: Fluorescence Activated Cell Sorting of Plant Protoplasts

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lipidvesikel-vermittelte Affinitätschromatographie unter Verwendung von Magnetic Activated Cell Sorting (LIMACS): Eine neue Methode zur Protein-Lipid-Wechselwirkungen Analyze

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Forschung

Biologie

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) Labeling und anschließende Fluorescence Activated Cell Sorting für Kultur-unabhängige Identifizierung von gelöstem organischem Kohlenstoff abbauenden Bakterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Zellspezifische Analyse<em&gt; Arabidopsis</em&gt; Blätter mit Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Glycan Profiling of Plant Cell Wall Polymers mit Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit Cell-Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Isolation von Normal und Krebs-assoziierte Fibroblasten von Fresh Gewebe durch Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Protein-Protein-Wechselwirkungen durch Bimolekulare Fluoreszenz-Komplementation in Tabak-Protoplasten-und Blätter-Visualisierte

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Messung des osmotischen Wasserdurchlässigkeit Koeffizient (P<sub&gt; F</sub&gt;) Von sphärischen Zellen: Isolierte Pflanzenprotoplasten als Beispiel

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Schnelle Analyse von Circadian Phänotypen in Arabidopsis Protoplasten Transfizierte mit einem lumineszenten Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizing Stromulus Frequenz mit Fluoreszenzmikroskopie

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...