Name: quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts
Uploaded: 2012-08-27T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

Diese Arbeit wurde vom Stiftungsfonds von der West Virginia University Mary Babb Randolph Cancer Center unterstützt. Wir danken Susette Mueller (Georgetown University) und Laura Kelley für die frühe Beratung und Unterstützung. Die Verwendung der West Virginia University Microscopy Imaging Facility (unterstützt von der Mary Babb Randolph Cancer Center und NIH Zuschüsse P20 RR16440, P30 und P30 RR032138 GM103488) wird dankbar anerkannt.

Ein. Produktion von Oregon Green 488-Gelatine beschichtete Deckgläser <ol><li> Planen eines unmarkierten 5% (w / w) stock Gelatine / Saccharose-Lösung durch Zugabe von 1,25 g Gelatine und 1,25 g Saccharose in PBS zu einem Endvolumen von 50 ml. Wärmen die Lager Gelatinelösung bis 37 ° C und sicherzustellen, dass es vor der Verwendung vollständig geschmolzen. Speichern die Endmischung bei 4 ° C. </li><li> Reinigen 13 mm Durchmesser # 1 Deckgläschen, indem ein einzelner Deckglas in jedes Well einer 24-Well-Kunsts...

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Die Fähigkeit, Zellen zu einer Verschlechterung der extrazellulären Matrix visualisieren hat die Entdeckung der molekularen Mechanismen in den frühen Schritten Zellinvasion beschäftigt unterstützt. Pionierarbeit von Wen-Tien Chen in den frühen 1980er Jahren 4,14,15 hat Beschichtung fluoreszenzmarkierten extrazelluläre Proteine ​​auf Deckgläsern für die anschließende mikroskopische Analyse als primäre Technik bei der Beurteilung invadopodia Funktion in einem breiten Spektrum von Zelltypen entsta...

<table border="1"><tbody><tr><td> Name des Reagenz / Instrument </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Gelatine </td><td> Sigma </td><td> G1890 </td><td> Schweinehaut </td></tr><tr><td> Saccharose </td><td> Fischer </td><td> BP220 </td><td></td></tr><tr><td> 12 mm-Deckgläschen </td><td> Fischer </td><td> 50-121-5159 </td><td></td></tr><tr><td> Platten mit 24 Vertiefungen </td><td> Fischer </td><td> 08-772-1 </td><td> BD Falcon </td></tr><tr><td> Salpetersäure </td><td> Ricca </td><td> R5326000 </td><td> 20% ige Lösung </td></tr><tr><td> Poly-L-Lysin </td><td> Electron Microscopy Sciences </td><td> 19.320-B </td><td> 0,1% ige Lösung </td></tr><tr><td> Glutaraldehyd </td><td> Sigma </td><td> G7526 </td><td> 8% ige Lösung </td></tr><tr><td&gt; Oregon Green 488-konjugierten Gelatine </td><td> Invitrogen </td><td> G-13186 </td><td></td></tr><tr><td> Natriumborhydrid </td><td> Sigma </td><td> 213462 </td><td></td></tr><tr><td> Triton X-100 </td><td> Fischer </td><td> BP151 </td><td></td></tr><tr><td> Rhodamin-Phalloidin </td><td> Invitrogen </td><td> R415 </td><td></td></tr><tr><td> Anti-Cortactin (Klon 4F11) </td><td> Millipore </td><td> 05-180 </td><td></td></tr><tr><td> Anti-FLAG-Antikörper </td><td> Millipore </td><td> MAB3118 </td><td></td></tr><tr><td> Alexa Fluor 647 Ziege anti-Maus-IgG </td><td> Invitrogen </td><td> A21235 </td><td></td></tr><tr><td> ProLong Gold-antifade </td><td> Invitrogen </td><td> P36930 </td><td></td></tr><tr><td> LSM 510 Konfokale Mikroskop </td><td> Zeiss </td><td></td><td></td></tr><tr><td> ImageJ Software </td><td> Public Domain </td><td></td><td> <a href="http://www.macbiophotonics.ca/imagej/" target="_blank">http://www.macbiophotonics.ca/imagej/</a> </td></tr><tr><td> AutoQuant X2.2 Software </td><td> Media Cybernetics </td><td></td><td></td></tr><tr><td> NIS Elements Software </td><td> Nikon </td><td></td><td></td></tr></tbody></table>

quantitative measurement of invadopodia-mediated extracellular matrix proteolysis in single and multicellular contexts

Cellular Invasion in lokalen Gewebe ist ein Prozess wichtig in der Entwicklung und Homöostase. Malregulated Invasion und anschließende Zellbewegung ist charakteristisch für mehrere pathologische Prozesse, einschließlich Entzündung, kardiovaskulärer Erkrankung und Tumorzellenmetastase ein. Focalized proteolytischen Abbau der extrazellulären Matrix (ECM)-Komponenten in der epithelialen oder endothelialen Basalmembran ist ein kritischer Schritt bei der Einleitung Zellinvasion. In Tumorzellen hat umfangreiche in vitro-Analyse bestimmt, dass ECM Abbau durch ventralen Aktin Membranplattformen vorstehenden Strukturen bezeichnet invadopodia 2,3 bewerkstelligt wird. Invadopodia Form in engen Apposition zu dem ECM, wo sie mäßig ECM Durchschlag durch die Wirkung von Matrixmetalloproteinasen (MMPs). Die Fähigkeit von Tumorzellen gegenüber invadopodia bilden direkt korreliert mit der Fähigkeit, sich in lokalen Stroma und assoziierten vaskulären Bauteile 3 einzudringen. _CONTENT &quot;&gt; Visualisierung invadopodia-vermittelten Abbau von ECM Zellen durch Fluoreszenzmikroskopie unter Verwendung von Farbstoff-markierten Proteine ​​Matrix auf Glas-Deckgläschen beschichtet wurde als die am weitesten verbreitete Technik zum Auswerten des Grades der zellulären Matrix Proteolyse und invasive Potential 4,5 entstanden. Hier beschreiben wir eine Version des Standard-Methode zur Erzeugung fluoreszenzmarkierten Glasdeckgläser Verwendung eines kommerziell erhältlichen Oregon Green 488-Gelatine-Konjugat. Dieses Verfahren ist einfach skaliert rasch bringen zahlreiche beschichtete Deckgläser. Wir zeigen einige der üblichen mikroskopischen Artefakte, die häufig auftreten Während dieses Verfahrens und wie diese vermieden werden. Schließlich haben wir standardisierten Methoden unter Verwendung leicht verfügbarer Computersoftware zur Quantifizierung von markierten Gelatinematrix Abbau durch einzelne Zellen und zellulären Populationen von gesamte vermittelt ermöglichen beschreiben. Die beschriebenen Verfahren bieten die Möglichkeit, genau und reproduzierbar zu überwachen invadopodiaAktivität, und kann auch als Plattform zur Bewertung der Wirksamkeit des Modulierens Proteinexpression oder Testen von Anti-invasiver Verbindungen auf extrazelluläre Matrix Abbau in Einzel-und mehrzellige Einstellungen dienen.

Watch this Scientific Journal Video about Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts at JoVE.com

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Wir beschreiben die prototypische Verfahren zur Herstellung Mikroskop Deckgläser mit fluoreszierenden Gelatine zur Visualisierung invadopodia-vermittelte Matrixabbau beschichtet. Rechenverfahren Verwendung verfügbarer Software für die Quantifizierung der resultierenden Ebenen der Matrix Proteolyse durch einzelne Zellen innerhalb einer Mischpopulation und für Gruppen, die vielzelligen gesamte mikroskopische Felder dargestellt.

Quantitative Messung der Invadopodia-vermittelte extrazellulären Matrix Proteolyse in Einzel-und Multizelluläre Contexts

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

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