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

Vi er takknemlige til Ken Iwata (OSI Pharmaceuticals, New York, USA) for reagenser og Fred Miller (Barbara Ann Karmanos Cancer Intitute, Detroit, USA) for cellen linjer.

1. Utarbeidelse av methocel løsning (500 ml) <ol><li> Autoclave 6 g methylcellulose i en 500 ml flaske som inneholder en magnetrører. </li><li> Oppløs methylcellulose i 250 ml forvarmet DMEM/F12 uten serum (60 ° C) i 20 min. </li><li> Tilsett 250 ml DMEM/F12 (RT) inneholder 10% hest serum. Mix natten (4 ° C). </li><li> Fjern løsningen ved sentrifugering (5000g, 2t, 4 ° C). </li><li> Ta den klare svært viskøs løsning til en ny tube (ca. 90-95% av stamløsningen). </li><li> Oppbevares ved 4 ° C før bruk. </l...

<ol>
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Vi etablerte en Spheroid modell der MCF10A1 celler og dets ondartet derivater invadere inn en kollagen matrise i en TGF-β-avhengig måte. Først er spheroids dannet i 96-brønner rund bunn suspensjon plater i nærvær av methylcellulose. Selvfølgelig kan også U-formet poly-HEMA belagte plater brukes til dette formålet. Methylcellulose er absolutt nødvendig i dette trinnet, fordi cellene vil dø i fravær av methylcellulose. TGF-β er lagt direkte til kollagen-methocel blanding siden vi fant ut at cellene respondert...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfritt) </td></tr><tr><td> Methyl cellulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avansert Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> pH indikator strimler </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-godt suspensjon plater </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96-godt feste TC plater </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 har motstridende roller i brystkreft progresjon ved å fungere som en tumor suppressor i den innledende fasen, men stimulerende invasjon og metastasering ved senere stadium 1,2. Videre er TGF-β ofte overexpressed i brystkreft og dens uttrykk korrelerer med dårlig prognose og metastasering 3,4. Mekanismer som TGF-β induserer invasjonen ikke er godt forstått. TGF-β utløser dens cellulære responser via TGF-β type II (TβRII) og type I (TβRI) reseptorer. Ved TGF-β-indusert heteromeric kompleks formasjon, phosphorylates TβRII den TβRI. De aktiverte TβRI innleder sitt intracellulære kanoniske signalveien ved phosphorylating reseptor Smads (R-Smads), dvs. Smad2 og Smad3. Disse aktiveres R-Smads skjema heteromeric komplekser med Smad4, som akkumuleres i kjernen og regulerer transkripsjon av målet gener 5. I tillegg til de tidligere described Smad veien, reseptor aktivering resulterer i aktivering av flere andre ikke-Smad signalveier, for eksempel mitogen Aktivert protein kinase (MAPK) veier 6. For å studere betydningen av TGF-β i ulike stadier av brystkreft, har vi gjort bruk av MCF10A celle system. Dette systemet består av spontant udødeliggjort MCF10A1 (M1) bryst epitelceller 7, forvandlet H-RAS M1-derivative MCF10AneoT (M2), som produserer premaligne lesjoner i mus 8, og M2-derivative MCF10CA1a (M4), som ble etablert fra M2 xenografts og former høy klasse karsinomer med evne til å metastasere til lungene 9. Denne MCF10A serien tilbyr muligheten til å studere svarene av celler med ulike graderinger av malignitet som ikke er forutinntatt av en annen genetisk bakgrunn. For analyse av TGF-β-indusert invasjon, genererte vi homotypic MCF10A Spheroid cellekulturer embedded i et 3D kollagen matrise in vitro (fig 1). Slike modeller ligner menneskelige svulster in vivo ved å etablere en gradient av oksygen og næringsstoffer, noe som resulterer i aktiv og invasive celler på utsiden og stillestående eller nekrotiske celler i innsiden av Spheroid 10. Spheroid baserte analyser har også vist seg å bedre rekapitulere legemiddelresistens enn monolayer kulturer 11. Denne MCF10 3D-modell system tillatt oss å undersøke virkningen av TGF-β signalering på invasive egenskaper av brystkreft celler i ulike stadier av malignitet.

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

En analyse for å kvantitativt måle Transforming Growth Factor (TGF)-β-indusert invasjonen i 3-dimensjonal kollagen gels er beskrevet. Denne analysen tar fordel av MCF10A rekke cellelinjer, som representerer ulike stadier av brystkreft utvikling. Denne metoden kan tas i bruk for å brukes med andre cellelinjer og kan brukes til å undersøke andre potensielle aktivatorer eller hemmere av invasjonen.

Spheroid Assay å måle TGF-β-indusert Invasion

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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