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

Dit werk werd ondersteund door fondsen uit schenkingen van de Universiteit van West Virginia Mary Babb Randolph Cancer Center. Wij danken Susette Mueller (Georgetown University) en Laura Kelley voor vroege advies en bijstand. Het gebruik van de Universiteit van West Virginia Microscopie Imaging Facility (ondersteund door de Mary Babb Randolph Cancer Center en, NIH subsidies P20 RR16440, P30 en P30 RR032138 GM103488) wordt dankbaar erkend.

1. Productie van Oregon Green 488-gelatine beklede dekglaasjes <ol><li> Bereid een ongelabelde 5% (w / w) stock gelatine / sucrose oplossing door toevoeging van 1,25 g gelatine en 1,25 g sucrose in PBS tot een eindvolume van 50 ml. Verwarm de stock gelatineoplossing tot 37 ° C en dat geschikt is volledig gesmolten voor gebruik. Bewaar het uiteindelijke mengsel bij 4 ° C. </li><li> Reinigen 13 mm diameter # 1 dekglaasjes door het plaatsen van een individuele dekglaasje in elk putje van een 24 well plastic weefselkwee...

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De mogelijkheid om cellen verslechtering van de extracellulaire matrix te visualiseren heeft bijgedragen tot het ontdekken van de moleculaire mechanismen die werkzaam zijn in de vroege stappen van celinvasie. Ontwikkeld door Wen-Tien Chen in de vroege jaren 1980 4,14,15, heeft coating fluorescent gelabelde extracellulaire eiwitten op dekglaasjes voor daaropvolgende microscopische analyse bleek de primaire techniek evaluatie invadopodia functie in een breed bereik aan celtypen. Het voorgeschreven toont de basi...

<table border="1"><tbody><tr><td> Naam van de Reagens / Instrument </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Reacties </td></tr><tr><td> gelatine </td><td> Sigma </td><td> G1890 </td><td> Varkens huid </td></tr><tr><td> sucrose </td><td> Visser </td><td> BP220 </td><td></td></tr><tr><td> 12 mm dekglaasjes </td><td> Visser </td><td> 50-121-5159 </td><td></td></tr><tr><td> Platen met 24 putjes </td><td> Visser </td><td> 08-772-1 </td><td> BD Falcon </td></tr><tr><td> salpeterzuur </td><td> Ricca </td><td> R5326000 </td><td> 20% oplossing </td></tr><tr><td> poly-L-lysine </td><td> Electron Microscopy Sciences </td><td> 19320-B </td><td> 0,1% oplossing </td></tr><tr><td> glutaaraldehyde </td><td> Sigma </td><td> G7526 </td><td> 8% oplossing </td></tr><tr><td&gt; Oregon Green 488-geconjugeerd gelatine </td><td> Invitrogen </td><td> G-13186 </td><td></td></tr><tr><td> natriumboorhydride </td><td> Sigma </td><td> 213462 </td><td></td></tr><tr><td> Triton X-100 </td><td> Visser </td><td> BP151 </td><td></td></tr><tr><td> rhodamine-phalloidin </td><td> Invitrogen </td><td> R415 </td><td></td></tr><tr><td> anti-cortactine (kloon 4F11) </td><td> Millipore </td><td> 05 tot 180 </td><td></td></tr><tr><td> Anti-FLAG antilichaam </td><td> Millipore </td><td> MAB3118 </td><td></td></tr><tr><td> Alexa Fluor 647 geit anti-muis 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 confocale microscoop </td><td> Zeiss </td><td></td><td></td></tr><tr><td> ImageJ software </td><td> Publiek domein </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 Cybernetica </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

Cellulaire invasie in de lokale weefsels is een proces van belang in de ontwikkeling en homeostase. Malregulated invasie en daaropvolgende cel beweging is kenmerkend meerdere pathologische processen, waaronder ontsteking, cardiovasculaire ziekte en tumorcel metastase 1. Gefocaliseerd proteolytische afbraak van extracellulaire matrix (ECM) in de epitheliale of endotheliale basaalmembraan is een kritische stap in de inleiding cellulaire invasie. In tumorcellen is uitgebreid in vitro analyse vastgesteld dat ECM afbraak wordt bereikt door ventrale actine-rijke membraan protrusive structuren genoemd invadopodia 2,3. Invadopodia vorm in nauwe appositie de ECM, waar ECM afbraak matigen door de werking van matrix metalloproteinases (MMP&#39;s). Het vermogen van tumorcellen om invadopodia vormen correleert direct met de mogelijkheid om binnen te dringen in lokale stroma en geassocieerde vasculaire componenten 3. _content &quot;&gt; Visualisatie van invadopodia-gemedieerde ECM afbraak van cellen door fluorescentiemicroscopie met kleurstof gemerkte eiwitten matrix aangebracht op dekglaasjes heeft zich ontwikkeld tot de meest voorkomende techniek voor het evalueren van de mate van proteolyse en cellulaire matrix invasieve potentieel 4,5. Hier beschrijven we een versie van de standaard werkwijze voor het fluorescentie-gemerkte dekglaasjes met behulp van een commercieel verkrijgbare Oregon Green-488 gelatine conjugaat. Deze methode is eenvoudig geschaald snel worden grote aantallen beklede dekglaasjes. We tonen enkele van de gemeenschappelijke microscopische artefacten die vaak optreden tijdens deze procedure en hoe deze kan worden vermeden. Tenslotte we gestandaardiseerde methoden met behulp van gemakkelijk verkrijgbare computersoftware voor kwantificering van gemerkt gelatine matrix afbraak gemedieerd door individuele cellen en gehele celpopulaties kunnen beschrijven. beschreven procedures de mogelijkheid bieden om nauwkeurig en reproduceerbaar te controleren invadopodiaactiviteit, en kan ook dienen als een platform voor het evalueren van de werkzaamheid van modulerende eiwitexpressie of testen van anti-invasieve verbindingen op extracellulaire matrix afbraak in enkele en meercellige instellingen.

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

We beschrijven de prototypische werkwijze voor het produceren microscoop dekglaasje bedekt met fluorescent gelatine visualiseren invadopodia-gemedieerde matrixafbraak. Rekentechnieken met beschikbare software gepresenteerd voor het kwantificeren van de niveaus van resulterende matrix proteolyse door enkele cellen in een gemengde populatie en voor meercellige groepen omvat gehele microscopische velden.

Kwantitatieve meting van Invadopodia-gemedieerde extracellulaire matrix proteolyse in Single en Meercellige contexten

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

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