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 taknemmelige for Ken Iwata (OSI Pharmaceuticals, New York, USA) til reagenser og Fred Miller (Barbara Ann Karmanos Cancer Intitute, Detroit, USA) for cellelinjer.

1. Forberedelse af methocel opløsning (500 ml) <ol><li> Autoklave 6 g methylcellulose i en 500 ml flaske med en magnetomrører. </li><li> Opløs methylcellulose i 250 ml forvarmet DMEM/F12 uden serum (60 ° C) i 20 min. </li><li> Tilsæt 250 ml DMEM/F12 (RT) indeholder 10% hest serum. Bland natten (4 ° C). </li><li> Ryd den løsning ved centrifugering (5000g, 2 timer, 4 ° C). </li><li> Tag den klare meget tyktflydende løsning på en ny slange (ca. 90-95% af bestanden løsning). </li><li> Opbevares ved 4 ° C indti...

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Vi har etableret en klumpformet model, hvor MCF10A1 celler og dens maligne derivater invaderer ind i en Kollagenmatrix&#39;en i en TGF-β-afhængig måde. For det første er spheroids dannet i 96-brønde rund bund suspension plader i overværelse af methylcellulose. Selvfølgelig kan også U-formet poly-HEMA plader anvendes til dette formål. Methylcellulose er absolut påkrævet i dette trin, da celler vil dø i mangel af methylcellulose. TGF-β er tilsat direkte til kollagen-methocel blanding, da vi fandt, at celler r...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer (valgfri) </td></tr><tr><td> Methylcellulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avanceret Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> pH indikator strips </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-samt suspension plader </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96-godt vedhæftning TC plader </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 modsatrettede roller i brystkræft progression ved at fungere som en tumor suppressor i den indledende fase, men stimulerende invasion og metastasering på et senere tidspunkt 1,2. Desuden er TGF-β ofte overudtrykt i brystkræft og dens udtryk korrelerer med dårlig prognose og metastasering 3,4. De mekanismer, som TGF-β fremkalder invasion er ikke godt forstået. TGF-β fremkalder dets celle-respons via TGF-β type II (TβRII) og type I (TβRI) receptorer. Ved TGF-β-induceret heteromeric kompleks dannelse, phosphorylates TβRII de TβRI. Den aktiverede TβRI indleder sit intracellulære kanoniske signalvejen fra phosphorylerende receptor Smads (R-Smads), dvs Smad2 og Smad3. Disse aktiveres R-Smads formular heteromeric komplekser med Smad4, som ophobes i kernen og regulere transskription af målet gener 5. Ud over de tidligere described Smad vej, receptor aktivering resulterer i aktivering af en række andre ikke-Smad signalveje, for eksempel mitogen aktiveret protein kinase (MAPK) veje 6. For at undersøge betydningen af ​​TGF-β i forskellige stadier af brystkræft, har vi gjort brug af MCF10A cellesystem. Dette system består af spontant udødeliggjort MCF10A1 (M1) bryst epitelceller 7, forvandlede H-RAS M1-derivat MCF10AneoT (M2), der producerer præmaligne læsioner i mus 8, og M2-afledte MCF10CA1a (M4), som blev etableret fra M2 implanteret og former høj kvalitet carcinomer med evnen til at metastaserer til lungerne 9. Denne MCF10A serie giver mulighed for at studere svarene af celler med forskellige kvaliteter af malignitet, der ikke er forudindtaget af en forskellig genetisk baggrund. Til analyse af TGF-β-induceret invasion, genererede vi homotypic MCF10A klumpformet cellekulturer embedded i en 3D Kollagenmatrix&#39;en in vitro (figur 1). Sådanne modeller ligner humane tumorer in vivo ved at etablere en gradient af ilt og næringsstoffer, hvilket resulterer i aktiv og invasive celler på ydersiden og passiv eller endda nekrotisk celler i den indvendige side af klumpformet 10. Klumpformet baserede analyser har også vist sig at bedre at rekapitulere resistens over for lægemidler end éncellelag kulturer 11. Denne MCF10 3D-model system gav os mulighed for at undersøge virkningen af ​​TGF-β signalering på invasive egenskaber bryst celler i forskellige stadier af 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 til kvantitativ måling Omdannelse vækstfaktor (TGF)-β-induceret invasion i 3-dimensionelle kollagen geler er beskrevet. Denne analyse tager fordel af MCF10A serie af cellelinier, der repræsenterer forskellige stadier af brystkræft udvikling. Denne metode kan vedtages skal bruges sammen med andre cellelinjer og kan bruges til at undersøge andre potentielle aktivatorer eller inhibitorer af invasion.

Klumpformet Assay til måling af TGF-β-induceret 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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