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

Dette arbeidet ble støttet av legat midler fra West Virginia University Mary Babb Randolph Cancer Center. Vi takker Susette Mueller (Georgetown University) og Laura Kelley for tidlig råd og hjelp. Bruken av West Virginia University Mikroskopi Imaging Facility (støttet av Mary Babb Randolph Cancer Center og NIH tilskudd P20 RR16440, P30 RR032138 og P30 GM103488) er takknemlig anerkjent.

1. Produksjon av Oregon Grønn 488-gelatin Coated Dekkglass <ol><li> Forbered en umerket 5% (w / w) lager gelatin / sukrose-løsning ved å tilsette 1,25 g gelatin og 1,25 g sukrose i PBS til et sluttvolum på 50 ml. Varm bestanden gelatinoppløsning til 37 ° C og at den er helt smeltet før bruk. Oppbevar endelige blandingen ved 4 ° C. </li><li> Rengjør 13 mm diameter # 1 glass dekkglass ved å plassere en individuell dekkglass i hver brønn av en 24 godt plastisk vevskulturplaten. Tilsett 500 ul av 20% salpetersy...

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Evnen til å visualisere celler nedverdigende den ekstracellulære matrix har hjulpet i å oppdage de molekylære mekanismene ansatt i de tidlige trinnene av celle invasjon. Utviklet av Wen-Tien Chen i tidlige 1980-tallet 4,14,15, har belegg fluorescensmerkede ekstracellulære proteiner på glass dekkglass for påfølgende mikroskopisk analyse dukket opp som den primære teknikk i å vurdere invadopodia funksjon over et bredt spekter av celletyper. Den foreskrevne protokollen demonstrerer grunnleggende metode...

<table border="1"><tbody><tr><td> Navn på Reagens / Instrument </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> gelatin </td><td> Sigma </td><td> G1890 </td><td> Porcine hud </td></tr><tr><td> sukrose </td><td> Fisher </td><td> BP220 </td><td></td></tr><tr><td> 12 mm dekkglass </td><td> Fisher </td><td> 50-121-5159 </td><td></td></tr><tr><td> 24 brønners plater </td><td> Fisher </td><td> 08-772-1 </td><td> BD Falcon </td></tr><tr><td> salpetersyre </td><td> Ricca </td><td> R5326000 </td><td> 20% løsning </td></tr><tr><td> poly-L-lysin </td><td> Elektronmikroskopi Sciences </td><td> 19320-B </td><td> 0,1% løsning </td></tr><tr><td> glutaraldehyd </td><td> Sigma </td><td> G7526 </td><td> 8% løsning </td></tr><tr><td&gt; Oregon Grønn 488-konjugert gelatin </td><td> Invitrogen </td><td> G-13 186 </td><td></td></tr><tr><td> natriumborhydrid </td><td> Sigma </td><td> 213462 </td><td></td></tr><tr><td> Triton X-100 </td><td> Fisher </td><td> BP151 </td><td></td></tr><tr><td> rhodamine-phalloidin </td><td> Invitrogen </td><td> R415 </td><td></td></tr><tr><td> anti-cortactin (klone 4F11) </td><td> Millipore </td><td> 05-180 </td><td></td></tr><tr><td> Anti-Flag antistoff </td><td> Millipore </td><td> MAB3118 </td><td></td></tr><tr><td> Alexa Fluor 647 Geit anti-mus IgG </td><td> Invitrogen </td><td> A21235 </td><td></td></tr><tr><td> Forleng Gold antifade </td><td> Invitrogen </td><td> P36930 </td><td></td></tr><tr><td> LSM 510 Confocal Microscope </td><td> Zeiss </td><td></td><td></td></tr><tr><td> ImageJ programvare </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 programvare </td><td> Media Cybernetics </td><td></td><td></td></tr><tr><td> NIS Elements programvare </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 invasjonen i lokale vev er en prosess viktig i utvikling og homeostase. Malregulated invasjon og påfølgende cellebevegelse er karakteristisk for flere patologiske prosesser, inkludert betennelse, kardiovaskulær sykdom og tumorcelle metastasering 1. Focalized proteolytisk nedbrytning av ekstracellulær matrix (ECM) komponenter i epiteliale eller endothelial basalmembran er et viktig skritt i å initiere cellulær invasjon. I kreftceller, har omfattende in vitro-analyse fastslått at ECM degradering oppnås ved ventrale aktin-rike membran protrusive strukturer betegnet invadopodia 2,3. Invadopodia skjema i nært apposisjon til ECM, hvor de modererer ECM sammenbrudd gjennom handling av matriksmetalloproteinaser (MMP). Muligheten av tumorceller til å danne invadopodia korrelerer direkte med evnen til å invadere i lokal stroma og tilhørende vaskulære komponenter 3. _content &quot;&gt; Visualisering av invadopodia-mediert ECM nedbrytning av celler ved fluorescerende mikroskopi bruker dye-merket matriksproteiner belagt på glass dekkglass har dukket opp som den mest utbredte teknikken for å vurdere graden av matrix proteolyse og cellulære invasiv potensial 4,5. Her beskriver vi en versjon av standard metode for generering fluorescently-merket glass dekkglass utnytte en kommersielt tilgjengelig Oregon Grønn-488 gelatin konjugat. Denne metoden er enkelt skaleres raskt produsere store mengder belagte dekkglass. Vi viser noen av de vanligste mikroskopiske gjenstander som ofte oppstår løpet av denne prosedyren, og hvordan disse kan unngås. Endelig beskriver vi standardiserte metoder ved bruk av lett tilgjengelig datamaskin programvare for å tillate kvantifisering av merket gelatin matrise nedbrytning mediert av individuelle celler og ved hele cellulære populasjoner. De beskrevne prosedyrer gir evnen til å nøyaktig og reproduserbart overvåke invadopodiaaktivitet, og kan også tjene som en plattform for å evaluere effekten av modulerende protein ekspresjon eller testing av anti-invasive forbindelser på ekstracellulær matriks nedbrytning i enkelt-og flercellede innstillinger.

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

Vi beskriver den prototypiske fremgangsmåte for fremstilling mikroskop dekkglass belagt med fluorescerende gelatin for å visualisere invadopodia-mediert matrise nedbrytning. Beregningsorientert teknikker ved hjelp av tilgjengelig programvare presenteres for å tallfeste resulterende nivåer av matrix proteolyse av enkeltceller i en blandet befolkning og for flercellede grupper som omfatter hele mikroskopiske felt.

Kvantitativ måling av Invadopodia-mediert Extracellular Matrix proteolyse i enkeltrom og Flercellede 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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