Os autores gostariam de agradecer as Dras. Rick Ryan, Umesh Patel, Jeff Till, Matthew Hsu, Jehangir Mistry e Michele Hatler de apoio durante o desenvolvimento desses projetos e protocolos.

Preparação de células: <ol><li> Antes da semeadura de células para o ensaio, a cultura de neurônios / astrócitos em meios de crescimento até confluentes ~ 70-80% (a menos de células semeadura diretamente do degelo ou isolamento). </li><li> Separar as células dos frascos de cultura / placas através do método apropriado para o tipo de células de interesse. Se necessário, os poços casaco ensaio de placa com poli-D-lisina ou proteína de matriz extracelular para melhorar a adesão celular. Co-culturas de astrócitos e neurônios pode ser semeado simultaneamente ou seqüencialmente. Para seedings seqüencial, chapeamento de astrócitos primeiro como uma camada de &quot;basal&quot; é recomendado, seguido por plaqueamento neuronal e diferenciação, se necessário. Ajustar a densidade de células, conforme apropriado para o tipo de célula, o tempo da cultura subseqüente, os parâmetros de interesse, etc Dependendo da idade da cultura, fonte de células e densidade de semeadura, culturas primárias podem variar muito em taxa de proliferação, GFAP expressão ou crescimento de neuritos - é importante caracterizar e otimizar seu sistema de célula para fornecer a relevância biológica mais para o seu modelo experimental, bem como para fornecer para geração de imagens eficaz, segmentação e análise usando HCS (ver Figura 1). Depois de adicionar células a placa (células podem ser semeadas em 90 mL de mídia, para facilitar o tratamento com toxina, como descrito no Passo 3 abaixo), permitir que a placa se sentar sobre uma superfície plana em temperatura ambiente por 15-30 min, permitindo distribuição uniforme de células. Após este período, as células se desenvolvem em meios de crescimento (37 ° C / 5% CO 2) por pelo menos 24 horas, em seguida, mudar as condições de cultura de diferenciação (por exemplo, soro baixo / NGF de PC12 células), conforme o caso. Continue cultura até atingir o nível desejado células de confluência ou diferenciação, mudando media em intervalos apropriados para o tipo de célula. </li><li> Tratamentos com células (compostos de controle, compostos de teste, etc) pode ser introduzido em qualquer momento durante este período da cultura, conforme apropriado para o tempo-curso de tratamento de interesse. Acrilamida, peróxido de hidrogênio e K-252 são fornecidas como compostos neurotóxicos controle. Reagentes suficientes são fornecidos para duplicar de 12 pontos curvas dose-resposta (incluindo uma dH 2 O DMSO ou de controle situado no dose-resposta) para todas as cinco placas de 96 poços. Os compostos são fornecidos em concentração 250X (para K-252a, assumindo um tratamento máximo de 1 mM) ou 10X (para acrilamida e peróxido de hidrogênio, supondo que tratamentos máximo de 100 mm e 10 mm, respectivamente). Preparação tratamento recomendado envolve meia-log (1: √ 10) diluição em série do composto 250X em DMSO, seguido de diluição em tampão de diluição Composto de 10X (compostos controle 10X originários de dH 2 O pode ser serialmente diluída diretamente em dH 2 O estéril ou tampão de diluição Compound). 10 L de cada tratamento podem então ser adicionados ao mL 90 de meios de cultura já está presente em cada poço, para uma concentração final de 1X (0,4% DMSO ou 10% dH 2 O). De dados de exemplo é fornecido por 24 ou 96 horas de tratamento composto de 37 ° C antes da fixação. </li></ol> Fixação de células e coloração de imunofluorescência: Nota: O tempo de coloração é de aproximadamente 2,5 horas pós-fixação. Não permitir que os poços a secar entre as etapas de coloração. Aspiração e dispensação de reagentes devem ser realizadas a taxas de fluxo baixo para diminuir a perda de células devido ao cisalhamento de fluidos. Todos os recomendados &quot;por bem&quot; volumes referem-se a um único poço de uma microplaca de 96 poços. Todos os recomendados &#39;por 96 poços da placa&#39; volumes incluem excesso de 25% as perdas de líquido manipulação de volume. Todas as etapas são realizadas coloração à temperatura ambiente (RT). Todos os tampões e soluções de anticorpos são estáveis ​​por pelo menos 24 horas à temperatura ambiente. <ol start="4"><li> No final do período de cultivo, pré-aquecido solução de fixação HCS (2X) a temperatura ambiente (RT) ou 37 º C, se desejar (12 placa mL/96-well). Em uma Cobertura de vapores químicos, adicionar 100 mL / poço diretamente para meios de cultura e permitir que a correção para 30 min em temperatura ambiente. Remover fixador / toxina contendo mídia e eliminar de acordo com os regulamentos para resíduos perigosos (ver MSDS). Se proceder imediatamente à coloração, lavar cada poço duas vezes com 200 mL de tampão de imunofluorescência HCS. Alternativamente, se as placas devem ser manchado em um momento posterior, lavar duas vezes com 200 mL de tampão de lavagem, enxágüe em seguida, deixar o volume segundo em poços e as placas loja hermeticamente fechado a 4 º C até coloração. </li><li> Se as amostras fixas foram armazenadas a 4 º C antes da coloração, lavar duas vezes com 200 mL de buffer HCS Imunofluorescência antes de prosseguir com protocolo de coloração. </li><li> Prepare solução de trabalho de Coelho Anti-βIII tubulina / Mouse Anticorpos Anti-GFAP HCS primária (6 placa mL/96-well) como segue: Adicionar 60 mL de cada anticorpo primário para 5,88 descongelado mL de tampão de imunofluorescência HCS. Misture bem. Remover o tampão de imunofluorescência anterior enxaguar. Adicionar 50 mL de solução de anticorpo primário a cada poço e incubar por 1 hora em temperatura ambiente. </li><li> Remover o anticorpo primáriosolução. Enxaguar três vezes com 200 mL de buffer de imunofluorescência HCS. </li><li> Preparar a solução de trabalho de HCS Anticorpo secundário / HCS Hoechst Nuclear Stain (6 placa mL/96-well) como segue: Adicionar 30 mL de cada anticorpo secundário descongelados e 30 mL de descongelado Hoechst HCS Nuclear Stain para 5,91 mL de tampão de imunofluorescência HCS. Misture bem, protegendo solução da luz. Remover anterior HCS buffer de imunofluorescência enxaguar. Adicionar 50 mL de HCS Anticorpo secundário / HCS Hoechst solução Nuclear Stain e incubar por 1 hora a RT, protegido da luz. </li><li> Remover HCS Secundária Antibody / Hoechst HCS solução Nuclear Stain. Enxágüe duas vezes com 200 mL de buffer de imunofluorescência HCS. </li><li> Remover anterior HCS buffer de imunofluorescência enxaguar. Enxágüe duas vezes com 200 mL de tampão de lavagem HCS, deixando o volume enxágüe segundo poços. </li><li> Placa de vedação e de imagem de imediato, armazenar ou placa a 4 º C protegido da luz até que esteja pronto para geração de imagens. </li></ol> Aquisição e Análise de Imagem: <ol start="12"><li> De imagem e análise de lâminas coradas pode ser realizada de uma variedade de plataformas disponíveis HCS, incluindo a IN Analyzer Cell (GE Healthcare), ArrayScan (ThermoFisher Científica) ou o Opera (Perkin Elmer). Algumas diretrizes para a imagem e análise são apresentadas na Tabela 1: </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222 Aquisição Diretrizes Imagem </td></tr><tr><td> Reagente de detecção </td><td> Lente objetiva </td><td> Faixa de excitação Filtro [pico / largura de banda (nm)] </td><td> Emissão Faixa de filtro [pico / largura de banda (nm)] </td></tr><tr><td> Hoechst HCS Nuclear Stain </td><td> 20X </td><td> 360/40 </td><td> 460/40 (ou 535/50, se necessário) </td></tr><tr><td> HCS Anticorpo secundário, FITC-Donkey anti-IgG de coelho </td><td> 20X </td><td> 480/40 </td><td> 535/50 </td></tr><tr><td> HCS Anticorpo secundário, Cy3-Donkey anti-rato IgG </td><td> 20X </td><td> 535/50 </td><td> 600/50 </td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222 Análise Diretrizes Imagem </td></tr><tr><td> Parâmetro celular </td><td> Detecção </td><td> Segmentação / Medição </td><td> Fundamentação </td></tr><tr><td> Número de células, características nucleares </td><td> Hoechst HCS Nuclear Stain </td><td> Região nuclear (460 canais de emissão nm). Contar o número de núcleos. Conteúdo de DNA (intensidade nuclear) ou análises área nuclear também são possíveis. </td><td> Use o número de células, características nucleares para determinar a perda de células, fenótipos toxicidade, etc Can &quot;filtro&quot; para aqueles núcleos associados βIII-tubulina ou expressão GFAP para obter em separado contagem de células neuronais e astrocitária / caracterizações (fortemente recomendado). </td></tr><tr><td> βIII-tubulina Expressão, crescimento de neuritos </td><td> HCS Anticorpo secundário, FITC-conjugado </td><td> Região citoplasmática (535 nm de emissão do canal). FITC sinal pode ser usado para distinguir os corpos celulares neuronais de neurites (por exemplo, através de áreas de célula mínima / média do corpo, comprimentos neurite mínimo / máximo e largura). Determinar parâmetros como o comprimento neurite total, contagem de neurite / celular, etc </td><td> Medições de crescimento de neuritos pode ser modulada durante a diferenciação neuronal ou como resultado de uma lesão química, estados de doença, etc, podem &quot;filtrar&quot; os corpos celulares para aqueles associados com βIII-tubulina expressão para obter a caracterização neuronal distintos (fortemente recomendado). </td></tr><tr><td> GFAP Expressão, Área astrocitária </td><td> HCS Anticorpo secundário, Cy3-conjugados </td><td> Região citoplasmática (600 canais de emissão nm). Cy3 sinal pode ser usado para definir a segmentação do citoplasma de astrócitos. Determinar parâmetros como a intensidade de sinal média citoplasmática, área da célula, etc </td><td> GFAP expressão e área de células astrócitos pode ser modulada durante o desenvolvimento de astrócitos ou como resultado de uma lesão química, estados de doença, etc, podem &quot;filtrar&quot; os corpos celulares para aqueles associados com a expressão GFAP astrocitária para obter a caracterização distintos (fortemente recomendado). </td></tr></tbody></table> . Tabela 1 Imagem Diretrizes de Aquisição e Análise - HCS222 βIII-Tubulin/GFAP Assay (Co-Cultura) Resultados representativos: <img src="/files/ftp_upload/1173/figure31173.png" alt="Figura 3" /> Figura 1. βIII-tubulina e GFAP immunofluorescence de rato misturado culturas de células neurais. Co-culturas de astrócitos do hipocampo de ratos primária com neurônios de rato, quer primária ou hipocampo de ratos células PC12 foram gerados por placas pré-astrócitos durante vários dias em cultura, seguido de semeadura neuronal. Neurônios primários foram cultivadas por mais 11 dias, enquanto PC12s foram cultivadas por mais dois dias sob condições de diferenciação (soro baixo / NGF). Manipulação de células fixação e imunomarcação foram realizadas de acordo com protocolos HCS222 ensaio. As células foram fotografadas no GE IN celular Analyzer 1000 (3.3) a 20X (neurônios primários) ou de ampliação objetiva de 10X (PC12) e analisados ​​com o GE IN celular Workstation 1000 Analyzer (3.4) crescimento de neuritos e algoritmos de análise multi-alvo Painel superior.: imagens fundidas de Hoechst HCS Nuclear Stain (azul), βIII-tubulina (verde) e GFAP fluorescência (vermelho). Análise em separado do canal de fluorescência βIII-tubulina permite a segmentação crescimento de neuritos (painel do meio: corpos celulares descritos no azul (neurônios primários) ou amarelo (PC12), neurites em verde (neurônios primários) ou azul claro (PC12)). Análise do canal de GFAP permite a segmentação de astrócitos (painel inferior: núcleos delineados em azul, o citoplasma em verde). GFAP segmentação para o primário do hipocampo de ratos neurônio / astrócitos co-cultura demonstra a capacidade de distinguir entre os núcleos / célula corpos que são GFAP (+) (verde contornos) e aqueles que são GFAP (-) (vermelho), permitindo a contagem de células separadas para neurônios e astrócitos em uma configuração de cultura mista. <img src="/files/ftp_upload/1173/figure41173.png" alt="Figura 4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="Figura 4 part2" /> Figura 2. Respostas dose de rato principal do hipocampo neurônio / astrócitos co-culturas a estresses tóxicos. Astrócitos do hipocampo de ratos primária (P6) foram semeadas em placas de 96 poços em meio de crescimento e cultivadas por 6 dias. Neurônios de rato principal do hipocampo (direto de descongelar) foram semeadas em cima do pré-plated astrócitos e cultivadas em meio de crescimento por 11 dias. As células foram tratadas com diluições seriadas de acrilamida ou peróxido de hidrogénio (máx. = concentração 100mM e 10mM, respectivamente) para as últimas 24 horas de cultura. Manipulação de células fixação e imunomarcação foram realizadas de acordo com protocolos HCS222 ensaio. As células foram fotografadas no GE IN celular Analyzer 1000 (3.3) a 20X (10 campos / poço) e analisadas (nuclear / cytoplasmic / neurite segmentação) usando o GE IN celular Workstation 1000 Analyzer (3.4) crescimento de neuritos e algoritmos de análise multi-alvo. Os dados apresentados são média ± EPM, n = 3. Vários parâmetros (crescimento de neuritos, intensidade GFAP, área de astrócitos e contagem de células neuronais ou astrocitária) foram investigados por dose-dependente tendências.

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M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

Os autores são todos os funcionários da Millipore Corporation.

Até o momento, experimentos in vitro concebido para estudo de avaliação do risco de neurotoxicidade utilizando culturas mistas de neurônios e astrócitos têm sido limitados. É bem aceito que as células gliais são essenciais na prestação de apoio espacial e metabólico para os neurônios, e desempenham um papel crítico na modulação neuronal várias funções, incluindo a migração neuronal, diferenciação e atividade sináptica. Astrócitos gliais também liberam citocinas e outros fatores solúveis que são capazes de induzir ambos os tolerância respostas adversas no tecido circundante neuronal, bem como promover neuronal de muitas toxinas. Demonstrando a profundidade da neuronal-glial interações, em co-cultura estudos, os astrócitos foram mostrados para proteger os neurônios contra a toxicidade do estresse oxidativo, enquanto comprometimento das funções astrocíticos, tais como manutenção de defesa antioxidante e os níveis de energia celular, tem se mostrado influenciam criticamente a sobrevivência neuronal. Estudos recentes têm sugerido que o uso de astrócitos em um sistema de teste in vitro-neurotoxicidade pode ser mais relevantes para a estrutura do sistema nervoso central humano e função de células neuronais sozinho. Apesar da crescente conscientização da importância fisiológica do neuronal-astrocíticos interações, tem sido difícil de executar análises quantitativas dessas interações em células intactas. O advento de Triagem de conteúdo de alta possibilita o desenvolvimento de novos testes poderosa para lidar com isso. Neste estudo, nós demonstramos que as análises quantitativas de vários fenótipos neuronais e astrocitária dentro de um único ensaio são possíveis por HCA. Em neurônios primários temos realizado medições quantitativas dos marcadores neurotoxicidade, como o número de neurônios, a contagem de neurite e comprimento neurite. Em astrócitos, gliose reativa é um biomarcador conhecido da neurotoxicidade, caracterizada por alterações na expressão de GFAP, o número de astrócitos e morfologia de astrócitos. Nós demonstramos que cada um destes parâmetros podem ser facilmente avaliada através de HCA. Estes estudos suportam o conceito de que HCA de neural específico biomarcadores poderiam ser usados ​​como parte de uma bateria de ensaios in vitro para tela rapidamente um grande número de produtos químicos para endpoints neurotóxicos. HCS222 Millipore do kit de análise de conteúdo de alta para o co-cultura de neurônios e astrócitos é composto de reagentes de alta qualidade e protocolos para a detecção simultânea de perfis βIII-tubulina e proteína fibrilar glial ácida (GFAP) em uma ampla variedade de modelos celulares de neurotoxicidade. Os reagentes altamente validado, desde que com este kit permite que o usuário para padronizar os ensaios, minimize-ensaio para ensaio-variabilidade, e reproduzível gerar imagens com uma relação sinal-background-alta.

<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rabbit Anti-&#x03B2;III-Tubulin HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201672</td>
<td>1 vial containing 300 &micro;L 
Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201649</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201671</td>
<td>1 vial containing 300 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200437</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hoechst HCS Nuclear Stain, 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200438</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Fixation Solution with Phenol Red, 2X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200434</td>
<td>1 bottle containing 100 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Immunofluorescence Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200435</td>
<td>1 bottle containing 1000 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Wash Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. 2007643</td>
<td>1 bottle containing 500 mL Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>Acrylamide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201679</td>
<td>1 vial containing 500 &micro;L of 1M acrylamide in dH2O Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hydrogen Peroxide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201730</td>
<td>1 vial containing 500 &micro;L of 100 mM hydrogen peroxide in dH2O Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>K-252a (Control Compound), 250X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201741</td>
<td>1 vial containing 100 &micro;L of 250 &micro;M K-252a in DMSO Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>DMSO for Compound Serial Dilution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200441</td>
<td>1 bottle containing 10 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Compound Dilution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200442</td>
<td>1 bottle containing 25 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Plate Sealers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200443</td>
<td>10 each Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>tissue culture-treated black/clear bottom microplates </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HCS imaging/analysis system</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

Alta Content Analysis (HCA) ensaios de combinar células e reagentes de detecção com imagem automatizada e poderosos algoritmos de análise de imagem, permitindo a medição de múltiplos fenótipos celulares dentro de um único ensaio. Neste estudo, utilizamos HCA para desenvolver um teste de novela para neurotoxicidade. Neurotoxicidade avaliação representa uma parte importante da avaliação de segurança de medicamentos, bem como sendo um foco significativo dos esforços de protecção ambiental. Além disso, neurotoxicidade é também um bem aceita<em&gt; In vitro</em&gt; Marcador do desenvolvimento de doenças neurodegenerativas, como Alzheimer e Parkinson. Recentemente, a aplicação de HCA para triagem neuronal tem sido relatada. Por rotulagem com células neuronais βIII-tubulina, ensaios de HCA pode proporcionar alto rendimento, não-subjetivo, medidas quantitativas de parâmetros como o número de neurônios, a contagem de neurite e comprimento dos neuritos, que podem indicar efeitos neurotóxicos. No entanto, o papel dos astrócitos permanece inexplorada nesses modelos. Astrócitos têm um papel fundamental na manutenção do sistema nervoso central (SNC) homeostase, e estão associados com as duas neuroproteção e neurodegradation quando elas são ativadas em resposta a substâncias tóxicas ou de estados de doença. GFAP é uma proteína de filamento intermediário expressa predominantemente nos astrócitos do SNC. Ativação astrocitária (gliose) leva à regulação alta de GFAP, comumente acompanhada de proliferação de astrócitos e hipertrofia. Este processo de gliose reativa tem sido proposta como um marcador precoce de danos ao sistema nervoso. O método tradicional para quantificação GFAP é por imunoensaio. Esta abordagem é limitada pela incapacidade de fornecer informações sobre a localização celular, morfologia e número de celular. Nós determinamos que o HCA pode ser usado para superar essas limitações e, simultaneamente, medir características múltiplos associados com gliose - mudanças na expressão GFAP, a hipertrofia de astrócitos, e proliferação de astrócitos - dentro de um único ensaio. Em co-cultura estudos, os astrócitos foram mostrados para proteger os neurônios contra vários tipos de insultos tóxicos e influenciam criticamente a sobrevivência neuronal. Estudos recentes têm sugerido que o uso de astrócitos em um<em&gt; In vitro</em&gt; Sistema de teste de neurotoxicidade pode ser mais relevantes para a estrutura e função do SNC humano do que as células neuronais sozinho. Assim, desenvolvemos um ensaio HCA para co-cultura de neurônios e astrócitos, composto por protocolos e validados, dirigidas a públicos específicos reagentes de detecção para perfil βIII-tubulina e glial fibrilar proteína ácida (GFAP). Este ensaio permite a análise simultânea de neurotoxicidade, crescimento de neuritos, gliose, morfologia neuronal e astrocitária e desenvolvimento neuronal e astrocitária em uma grande variedade de modelos de celulares, o que representa um romance, não-subjetiva, teste de alto rendimento para a avaliação de neurotoxicidade. O ensaio tem um grande potencial para a detecção aprimorada de neurotoxicidade e maior produtividade em pesquisa em neurociência e descoberta de drogas.

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Este artigo descreve um protocolo de romance e de reagente conjunto projetado para a medição sensível de efeitos neurotóxicos de compostos e tratamentos em co-culturas de neurônios e astrócitos usando análise de conteúdo de alta. Resultados demonstram que a análise de conteúdo de alta tecnologia representa um romance empolgante para a avaliação de neurotoxicidade.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

a-neuronal-astrocyte-co-culture-assay-for-high-content-analysis

Research

JoVE Journal

Biology

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Vídeo: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Culturas de células neuronais de Aplysia para imagens de alta resolução de Cones de Crescimento

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Biologia

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Um compartimento Multi-glia do SNC Neuron-Co-cultura plataforma microfluídica

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Análise de Schwann-astrócito Interações Usando In Vitro Ensaios

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantificação de dsDNA usando a Hitachi F-7000 Espectrofotômetro de Fluorescência e PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Medição Rendimento Absoluto Quantum of amostras de pó

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Um fluxo de trabalho de imagem de alta de conteúdo para estudar Grb2 Complexos de Sinalização por clonagem de expressão

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análise qPCRTag - A High Throughput, PCR em Tempo Real Ensaio para Sc2.0 Genotipagem

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Análise alto teor de triagem para avaliar os efeitos toxicológicos de Constituintes nocivas e potencialmente prejudiciais (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...