Name: spheroid assay to measure tgf-&#946;-induced invasion
Uploaded: 2011-11-16T13:00:00
Description: Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

We zijn dankbaar dat Ken Iwata (OSI Pharmaceuticals, New York, USA) voor de reagentia en Fred Miller (Barbara Ann Karmanos Kanker Intitute, Detroit, USA) voor de cellijnen.

1. Voorbereiding van de Methocel oplossing (500 ml) <ol><li> Autoclaaf 6 g methylcellulose in een 500 ml fles met een magneetroerder. </li><li> Los van de methylcellulose in 250 ml voorverwarmd DMEM/F12 zonder serum (60 ° C) gedurende 20 minuten. </li><li> Voeg 250 ml van DMEM/F12 (RT) met 10% paard serum. Mix &#39;s nachts (4 ° C). </li><li> Wis de oplossing door centrifugeren (5000g, 2h, 4 ° C). </li><li> Neem het heldere zeer viskeuze oplossing voor een nieuwe buis (ongeveer 90-95% van de voorraad-oplossing). </...

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We hebben een sferoïde model waarin MCF10A1 cellen en zijn kwaadaardige derivaten binnen te vallen in een collageen matrix in een TGF-β-afhankelijke manier. Eerst worden sferoïden gevormd in 96-wells ronde bodem schorsing platen in de aanwezigheid van methylcellulose. Natuurlijk kan ook de U-vormige poly-HEMA gecoate platen worden gebruikt voor dit doel. Methylcellulose is absoluut noodzakelijk in deze stap, aangezien cellen zullen sterven in de afwezigheid van methylcellulose. TGF-β wordt direct toegevoegd aan de c...

<table border="1"><tbody><tr><td> Naam van het reagens </td><td> Vennootschap </td><td> Catalogus nummer </td><td> Commentaar (optioneel) </td></tr><tr><td> Methylcellulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Geavanceerde BioMatrix </td><td> 5005-B </td><td></td></tr><tr><td> pH indicator strips </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-well schorsing platen </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96-well hechting TC platen </td><td> Greiner Bio-One </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β heeft tegengestelde rollen in borstkanker progressie door te fungeren als een tumor suppressor in de beginfase, maar het stimuleren van invasie en metastase in een later stadium 1,2. Bovendien is TGF-β vaak tot overexpressie gebracht in borstkanker en de uitdrukking ervan correleert met een slechte prognose en metastase 3,4. De mechanismen waarmee TGF-β induceert invasie niet goed begrepen. TGF-β lokt haar cellulaire reacties via TGF-β type II (TβRII) en type I (TβRI) receptoren. Bij de TGF-β-geïnduceerde heteromeric complex vorming, TβRII fosforyleert de TβRI. De geactiveerde TβRI initieert zijn intracellulaire canonieke signaalroute door fosforylatie receptor Smads (R-Smads), dat wil zeggen Smad2 en Smad3. Deze geactiveerde R-Smads vorm heteromeric complexen met Smad4, die zich ophopen in de kern en het reguleren van de transcriptie van doelwitgenen 5. In aanvulling op de eerder described Smad pad, receptor activering resulteert in de activering van een aantal andere niet-Smad signaalwegen, bijvoorbeeld Mitogeen Activated Protein Kinase (MAPK) wegen 6. De studie van de rol van TGF-β in verschillende stadia van borstkanker, hebben we gebruik gemaakt van de MCF10A cel systeem. Dit systeem bestaat uit spontaan vereeuwigd MCF10A1 (M1) borst epitheelcellen 7, de H-RAS getransformeerd M1-derivaat MCF10AneoT (M2), die premaligne letsels veroorzaakt bij muizen 8, en de M2-afgeleide MCF10CA1a (M4), dat werd vastgesteld op basis van M2 xenografts en vormen hoogwaardig carcinomen met de mogelijkheid om uitzaaien naar de longen 9. Dit MCF10A serie biedt de mogelijkheid om de reacties van cellen te bestuderen met verschillende graden van maligniteit die niet vertekend door een verschillende genetische achtergrond. Voor de analyse van TGF-β-geïnduceerde invasie genereerden wij homotypic MCF10A sferoïde celculturen embeT OEGEVOEGDE in een 3D-collageen-matrix in vitro (figuur 1). Dergelijke modellen sterk lijken op menselijke tumoren in vivo door de oprichting van een gradiënt van zuurstof en voedingsstoffen, wat resulteert in actieve en invasieve cellen aan de buitenkant en rustende of zelfs necrotische cellen in de binnenkant van de sferoïde 10. Sferoïde gebaseerde testen hebben ook aangetoond dat resistentie tegen geneesmiddelen beter samenvatten dan monolaagculturen 11. Dit MCF10 3D-model-systeem liet ons toe om de impact van de TGF-β signalering te onderzoeken op de invasieve eigenschappen van borst-cellen in verschillende stadia van maligniteit.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-ÃŽÂ²-induced Invasion at JoVE.com

Spheroid Assay to Measure TGF-ÃƒÅ½Ã‚Â²-induced Invasion

Een test om kwantitatief te meten Transforming Growth Factor (TGF)-β-geïnduceerde invasie in 3-dimensionale collageen gels is beschreven. Deze test maakt gebruik van de MCF10A reeks van cel-lijnen, die verschillende stadia van de ontwikkeling van borstkanker te vertegenwoordigen. Deze methode kan worden vastgesteld om te worden gebruikt met andere cellijnen en kan worden gebruikt om andere potentiële activatoren of inhibitoren van een invasie te onderzoeken.

Sferoïde Assay voor TGF-β-geïnduceerde Invasion Measure

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

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Spheroid Assay to Measure TGF-&#946;-induced Invasion

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