Name: isolation of cardiomyocyte nuclei from post-mortem tissue
Uploaded: 2012-07-10T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Nós gostamos de reconhecer Marcelo Toro para a assistência com a citometria de fluxo. Este estudo foi financiado pelo sueco Coração e Pulmão Foundation, da Comissão Européia, FP7 &quot;CardioCell&quot;, Sueco de Pesquisa do Conselho, seguros AFA e ALF. OB foi apoiado pelo Deutsche Forschungsgemeinschaft.

1. Isolamento dos Núcleos Cardiac <ol><li> Revestimento tubos de ultracentrifugação (Tubos centrífuga Beckman # 363664) com 10 ml de solução de revestimento de 1% BSA / PBS. Tapar os tubos e deixá-los girar durante 30 min num rotor tubo. Remover a solução de revestimento e deixar que o tubos de centrifugação de ar seco (um tubo por coração do rato é necessária para a análise de corações de rato individuais, alternadamente até 5 corações de rato ou 1 g de tecido do coração a partir de uma espéci...

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A identificação precisa de núcleos de cardiomiócitos é crucial para a análise dos processos regenerativos no miocárdio 2,3. As técnicas convencionais para isolar cardiomiócitos de tecido fresco baseiam-se principalmente a digestão enzimática de proteínas da matriz extracelular e da subsequente purificação a partir de células intersticiais por centrifugação a baixa velocidade. A purificação adicional de cardiomiócitos de vida a partir de células estaminais embrionárias (ESC) pode ser real...

<table border="1"><tbody><tr><td> 1. Tampão de Lise </td></tr><tr><td> Nome do reagente </td></tr><tr><td> 0,32 M de sacarose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de CaCl2 </td></tr><tr><td> 5 mM de acetato de magnésio </td></tr><tr><td> 2,0 mM de EDTA </td></tr><tr><td> 0,5 mM de EGTA </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Tampão de sacarose </td></tr><tr><td> Nome do reagente </td></tr><tr><td> 2,1 M de sacarose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de acetato de magnésio </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Tampão de núcleos de armazenamento (NSB plus) </td></tr><tr&gt; <td> Nome do reagente </td></tr><tr><td> 0,44 M de sacarose </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM de KCl </td></tr><tr><td> 10 mM MgCl2 </td></tr><tr><td> 1,5 mM de espermina </td></tr></tbody></table><table border="1"><tbody><tr><td> Reagentes e Equipamentos </td><td> Companhia </td></tr><tr><td> Isotipo IgG de coelho-CHIP Grau, # ab37415 </td><td> Abcam </td></tr><tr><td> De coelho anti-PCM-1 anticorpo, # HPA023374 </td><td> Anticorpos Atlas </td></tr><tr><td> Burro seg. anticorpo, anti-coelho Alexa 488 Fluor, # A-21.206 ou sec equivalente. anticorpo fluorescente </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> Os filtros de células de 30 uM, 70 uM e 100 uM </td><td> BD Biosciences </td></tr><tr><td> Vidro douncer (40 ml) epilão &quot;L&quot; </td><td> VWR (Wheaton Industries, Inc.) </td></tr><tr><td> T-25 homogeneizador Ultra-Turrax </td><td> IKA Alemanha </td></tr><tr><td> Dispersão ferramenta S25 N-18 G </td><td> IKA Alemanha </td></tr><tr><td> Centrífuga Beckman Avanti </td><td> Beckman Coulter </td></tr><tr><td> Tubos Falcon de 15 ml e 50 ml </td><td> VWR </td></tr><tr><td> Tubos de centrífuga Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotor livre balançando </td><td> Beckman Coulter </td></tr><tr><td> Citómetro Influx </td><td> Beckman Coulter </td></tr><tr><td> Rotator tubo </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificação de núcleos de cardiomiócitos tem sido um desafio em cortes de tecido como a maioria das estratégias de contar apenas com proteínas marcadoras citoplasmáticos 1. Eventos raros em miócitos cardíacos, tais como a proliferação e apoptose necessitam de uma identificação precisa dos núcleos dos miócitos cardíacos para analisar a renovação celular e na homeostasia em condições patológicas 2. Aqui, nós proporcionar um método para isolar a partir de núcleos de cardiomiócitos pós tecido mortem por sedimentação densidade e imunomarcação com anticorpos contra o material pericentriolar 1 (PCM-1) e triagem de citometria de fluxo posterior. Esta estratégia permite uma análise com rendimento elevado e de isolamento, com a vantagem de se trabalhar igualmente bem em tecido fresco e material de arquivo congelado. Isto torna possível estudar o material já recolhidos na biobancos. Esta técnica é aplicável e testado em uma vasta gama de espécies e adequados para várias aplicações tais como a jusante de carbono-14 namoro 3, a célula-cycle análise 4, visualização de análogos da timidina (por exemplo, BrdU e UDI) 4, a análise de transcriptoma e epigenética.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Núcleos cardíaca são isolados através de sedimentação densidade e immunolabeled com anticorpos contra o material pericentriolar 1 (PCM-1) para identificar e classificar os núcleos dos cardiomiócitos por citometria de fluxo.

Isolamento dos núcleos dos cardiomiócitos a partir de Post-mortem de tecidos

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

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