Name: screening assay for oxidative stress in a feline astrocyte cell line, g355-5
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1. Cultura de células de astrócitos felino e tratamento com H 2 O 2 <ol><li> Linha de cultura de células G355-5 em um balão de 75 cm 2 com a mídia DMEM acrescido de nutrientes, a 37 ° C e 5% de CO 2 até confluentes 60% (~ 5 dias). Substituir a mídia a cada 1-2 dias ou quando necessário para manter uma cultura saudável. </li><li> Remova a mídia e adicionar 2 ml de tripsina 0,25%. Levante as células por pipetagem por 45 s - 1 min à temperatura ambiente (RT). </li>...

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Um mecanismo muitas vezes sugerido de vírus danos induzidos neuronal é o estresse oxidativo 1, 4, 7, 9, 13, 15, 16, 18-22, 27, 31. Basicamente, propõe-se que através da exposição viral da glia (astrócitos e microglia) liberação de radicais de oxigênio reativo, tais como, hidróxido, ânion superóxido, óxido nítrico, e / ou peróxido de hidrogênio, que são tóxicos para os neurônios. Da mesma forma, o estresse oxidativo também tem sido sugerido como um mecanismo patológico em grandes metanfet...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> DMEM </td><td> Cellgro </td><td> 15-013-CV </td><td></td></tr><tr><td> MEM Vitaminas </td><td> Cellgro </td><td> 25-020-cl </td><td> 1% (v / v) </td></tr><tr><td> L-glutamina </td><td> Hyclone </td><td> 17-605E </td><td> 1% (v / v) </td></tr><tr><td> Não Aminoácidos Essenciais </td><td> Hyclone </td><td> 25-025-cl </td><td> 1% (v / v) </td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.03 </td><td> 20% (v / v) </td></tr><tr><td> Aminoácidos Essenciais </td><td> Cellgro </td><td> 25-030-cl </td><td> 0,2% (v / v) </td></tr><tr><td> Sistema de filtragem a vácuo 500ml </td><td> VWR </td><td> 87006-076 </td><td></td></tr><tr><td> 15 ml tubos cônicos </td><td> Falcão </td><td> 352097 </td><td></td></tr><tr><td> 75 centímetros de cultura de tecidos 2 frascos </td><td> Falcão </td><td> 353136 </td><td></td></tr><tr><td> 6 placa de cultura de tecido bem </td><td> Falcão </td><td> 353224 </td><td></td></tr><tr><td> CM-H 2 DCFDA </td><td> Invitrogen </td><td> C6827 </td><td></td></tr><tr><td> Iodeto de propídio (PI) </td><td> Sigma </td><td> P4170-10mg </td><td></td></tr><tr><td> Tripsina </td><td> Lonza </td><td> 17-160F </td><td></td></tr><tr><td> H 2 O 2 </td><td> Sigma </td><td> H6520 </td><td></td></tr><tr><td> Antibiótico HyQ </td><td> Hyclone </td><td> SV30079.01 </td><td> 0,1% (v / v) </td></tr><tr><td> G355-5 células </td><td> ATCC </td><td> CRL-2033 </td><td> Cérebro felino normal </td></tr></tbody></table>

screening assay for oxidative stress in a feline astrocyte cell line, g355-5

Um mecanismo muitas vezes sugerido de vírus danos induzidos neuronal é o estresse oxidativo. Astrócitos têm um papel importante no controle do estresse oxidativo do Sistema Nervoso Central (SNC). Astrócitos ajudar a manter um ambiente homeostático para os neurônios, assim como proteger os neurônios de Espécies Reativas de Oxigênio (ROS). CM-H2DCFDA é um indicador de células-permeável para a presença de ROS. CM-H 2 DCFDA entra na célula como um composto não-fluorescente, e se torna fluorescente após esterases celulares remover os grupos acetato, eo composto é oxidado. O número de células, medido por citometria de fluxo, que são encontrados para ser fluorescentes verde é uma indicação do número de células que estão em um estado oxidativo. CM-H 2 DCFDA é suscetível à oxidação por um grande número de ROS diferente. Esta falta de especificidade, considerando que ROS pode oxidar CM-H 2 DCFDA, faz deste composto um regente valiosa para uso nas fases iniciais de uma investigação patogênese, como este teste pode ser usado para a tela para um ambiente oxidativo celular, independentemente do que o oxigênio radical ou combinação de ROS são responsáveis ​​pelas condições celular. Uma vez que foi estabelecido que ROS estão presentes por oxidação de DCFDA 2 CM-H, em seguida, experimentos adicionais podem ser realizados para determinar qual ROS ou combinação de EROs estão envolvidas no processo de patogênese particular. Os resultados deste estudo demonstram que com a adição de peróxido de hidrogênio em um aumento DCFDA 2 CM-H fluorescência foi detectada em relação aos controles salina, indicando que este teste é um teste útil para a detecção de um ambiente oxidativo dentro G355-5 células, uma linhagem celular felina astrócitos.

Watch this Scientific Journal Video about Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5 at JoVE.com

Screening Assay for Oxidative Stress in a Feline Astrocyte Cell Line, G355-5

Um método de triagem para detectar ambientes oxidativo celular é medir a oxidação do CM-H2DCFDA. Uma vez oxidada dentro de uma célula, CM-H2DCFDA muda de não-fluorescentes em um composto fluorescente. Esta mudança de fluorescência é medida por citometria de fluxo e indica o número de células em um ambiente oxidativo.

Ensaio de triagem para Estresse Oxidativo em uma linhagem de células Feline astrocitária, G355-5

An often-suggested mechanism of virus induced neuronal damage is oxidative stress. Astrocytes have an important role in controlling oxidative stress of ...

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