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

Somos gratos a Ken Iwata (OSI Pharmaceuticals, New York, EUA) para reagentes e Miller Fred (Barbara Ann Karmanos Cancer Intitute, Detroit, EUA) para as linhas de celular.

1. Preparação da solução Methocel (500 mL) <ol><li> Autoclave g 6 de metilcelulose em uma garrafa de 500 ml contendo um agitador magnético. </li><li> Dissolver a metilcelulose em 250 ml de DMEM/F12 pré-aquecido sem soro (60 ° C) por 20 min. </li><li> Adicione 250 ml de DMEM/F12 (RT), contendo soro de cavalo 10%. Misturassem durante a noite (4 ° C). </li><li> Clara a solução por centrifugação (5000g, 2h, 4 ° C). </li><li> Tome a solução clara e altamente viscoso para um novo tubo (cerca de 90-95% da solu...

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Nós estabelecemos um modelo esferóide em que MCF10A1 células e seus derivados malignos invadem em uma matriz de colágeno de uma forma TGF-β-dependente. Primeiro, esferóides são formados em placas redondas de 96 poços de fundo de suspensão na presença de metilcelulose. Naturalmente, também em forma de U poli-HEMA placas revestidas podem ser usados ​​para esta finalidade. Metilcelulose é absolutamente necessário nesta etapa, já que as células morrerão na ausência de metilcelulose. TGF-β é adicionado...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> Metil celulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avançados Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> indicador de pH tiras </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> De 96 poços de suspensão placas </td><td> Greiner Bio-one </td><td> 650 185 </td><td></td></tr><tr><td> De 96 poços de adesão TC placas </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 tem papéis opostos na progressão do câncer de mama, agindo como um supressor de tumor na fase inicial, mas estimulando a invasão e metástase na tarde 1,2 palco. Além disso, o TGF-β é freqüentemente overexpressed no câncer de mama e sua expressão se correlaciona com o prognóstico pobre e metástases 3,4. Os mecanismos pelos quais TGF-β induz a invasão não são bem compreendidos. TGF-β provoca suas reações celulares via TGF-β tipo II (TβRII) e eu tipo (TβRI) receptores. TGF-β sobre induzida por formação heteromeric complexo, TβRII fosforila o TβRI. O TβRI ativada inicia a sua via de sinalização intracelular canônica pela Smads receptor fosforilar (R-Smads), ou seja Smad2 e Smad3. Estes ativados R-Smads formam complexos com heteromeric Smad4, que se acumulam no núcleo e regulam a transcrição de genes-alvo 5. Além do anteriormente dvia Smad escribed, os resultados receptor de ativação na ativação de diversas outras organizações não-Smad vias de sinalização, por exemplo Mitógeno proteína quinase ativada (MAPK) Vias 6. Para estudar o papel da TGF-β em diferentes estágios do câncer de mama, fizemos uso do sistema de célula MCF10A. Este sistema é composto de células espontaneamente imortalizado MCF10A1 (M1) de mama epiteliais 7, o H-RAS transformou M1 derivados MCF10AneoT (M2), que produz lesões pré-malignas em camundongos 8, eo MCF10CA1a M2 derivados (M4), que foi estabelecida a partir de xenoenxertos M2 e os formulários de alto grau de carcinomas com a capacidade de metástase para o pulmão 9. Esta série MCF10A oferece a possibilidade de estudar as respostas de células com diferentes graus de malignidade que não são influenciados por uma base genética diferente. Para a análise de TGF-β induzida por invasão, geramos homotypic MCF10A esferóide celular embe culturasdded em uma matriz de colágeno 3D in vitro (Fig. 1). Tais modelos se assemelham tumores humanos in vivo, estabelecendo um gradiente de oxigênio e nutrientes, resultando em células ativas e invasiva sobre as células fora e quiescente ou mesmo necrose no interior dos 10 esferóide. Esferóide ensaios com base também foram mostrados para melhor recapitular resistência às drogas do que as culturas em monocamada 11. Este sistema MCF10 modelo 3D permite estudar o impacto do TGF-β sinalização nas propriedades invasivas das células de mama em estágios diferentes de malignidade.

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

Um ensaio para medir quantitativamente Transforming Growth Factor (TGF)-β induzida por invasão em gel 3-dimensional colágeno é descrito. Este ensaio se aproveita da série MCF10A de linhas de células, que representam diferentes fases de desenvolvimento de câncer de mama. Este método pode ser adotado para ser usado com outras linhagens de células e pode ser usado para investigar outras possíveis ativadores ou inibidores de invasão.

Ensaio esferóide para TGF-β Medida induzida 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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