Os autores gostariam de agradecer a John Woolford para a base inicial deste método, TGK é suportado pelo NIH e GM057483 AI076245 e RPC é apoiada por GM77073.

1. Preparação de gradientes de sacarose 7-47% <ol><li> Prepare gradientes de sacarose um dia antes do uso para permitir gradientes para tornar-se contínuo. Tornar estéril 7% e soluções de sacarose 47% contendo 50 mM NH 4 Cl, 50 mM Tris pH 7,0 Acetato e MgCl 2 12mm e armazenar a 4 ° C 1. </li><li> Por 6 gradientes misturar os 7% e as soluções estoque 47% de sacarose para fazer 48 mL de cada uma das seguintes concentrações de sacarose. Adicionar TDT (1M estoque) para cada solução de sacarose a uma concentração final de 1mM. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Concentração Final </td><td> Sacarose 7% </td><td> 47% de sacarose </td></tr><tr><td> 7% </td><td> 48 mL </td><td> 0 </td></tr><tr><td> 17% </td><td> 36 mL </td><td> 12 mL </td></tr><tr><td> 27% </td><td> 24 mL </td><td> 24 mL </td></tr><tr><td> 37% </td><td> 12 mL </td><td> 36 mL </td></tr><tr><td> 47% </td><td> 0 </td><td> 48 mL </td></tr></tbody></table></li><li> , A fim de derramar os gradientes, anexar uma pipeta Pasteur a uma seringa de 20 mL de segurança e vedação com Parafilm ou usar uma agulha longa. Adicionar 7 ml de sacarose 7% para o fundo de um tubo de 1 x 3,5 ultracentrífuga &quot;polyallomer. Em seguida adicione 7 mL da solução de sacarose 17% sob a solução de 7%, colocando a ponta da pipeta Pasteur perto fundo do tubo e pipetar não mais do que 0,3 mL / sec Repetir. com 7 mL cada de 27%, 37% e soluções de sacarose 47%. Você deve observar linhas claras entre as camadas, indicando que a mistura mínima ocorreu. loja gradientes a 4 ° C durante a noite junto com os rotores, garrafas e tubos que serão utilizados na colheita de amostras. </li></ol> 2. Preparação do extrato de levedura <ol><li> Crescer 125 mL de cultura de levedura para um OD 600 de 0,8-1,0. Adicionar cicloheximida para a cultura a uma concentração final de 100 mcg / mL e continuar apertando a 30 ° C por 15 min. </li><li> Enquanto isso, prepare tampão de lise contendo 80 mcg / mL cicloheximida, 200 heparina mcg / mL, DEPC 0,2%, e 10 mM Tris-HCl pH 7,5, 0,1 M NaCl, 30mm MgCl 2. Mantenha tudo no gelo a partir deste ponto. </li><li> Despeje a cultura em um frasco de centrífuga de 250 mL e encha com gelo. Centrifugar a 8000 xg (7.250 rpm em um rotor SLA-1500) por 5 min a 4 ° C. Lave o pellet uma vez com 10 mL de tampão de lise de gelo frio e transferência para tubos de 15 mL de polipropileno. Centrifugar a 5900 xg (~ 6.000 rpm em um rotor SLA-600TC) por 3 min a 4 ° C. Ressuspender o sedimento em 0,5 mL de transferência de tampão de lise, para tubo de 1,5 ml e adicionar 0,5 mL de refrigerados, cozido contas ácido lavado vidro. Vortex as células para quatro intervalos de 30 segundos, resfriando o tubo em gelo durante 30 segundos entre cada intervalo. Adicionar uma outra de 0,5 mL de tampão de lise no tubo de 1,5 mL. Centrifugar a velocidade máxima em microcentrífuga (14.000 rpm em uma microcentrífuga Eppendorf 5417R) por 10 min a 4 ° C. Transfira o sobrenadante para um novo tubo de microcentrífuga de 1,5 mL. Retire 10 ml para um tubo separado para determinar a OD 260 / OD 280 ratio (veja o passo 3.1). Extratos armazenar a -80 ° C. </li></ol> 3. Preparação de extratos de células de mamíferos <ol><li> Para células aderentes, crescer em 100 prato mm a confluência ~ 70%. Você vai precisar de um prato de 100 mm por 11 gradiente ml (rendimento típico é ~ 20 OD 260 unidades). Escala a quantidade linearmente para tamanhos gradiente maior ou menor. Para as células não aderentes, cerca de 1 x 10 7 células são necessárias por 11 ml de gradiente. </li><li> Antes de lise, adicione cicloheximida a 100 mcg / mL. Incubar a 37 ° C por 15 min. Transferência de placas de gelo e lave as células duas vezes em PBS gelado. Todos os passos futuros deve ser realizado em 0-4 ° C. </li><li> Remover todos os vestígios de PBS por aspiração (vamos placas de drenagem em uma cama em ângulo de gelo) e adicionar 1 mL tampão de lise, raspar e transferência para pré-refrigerados tubo de 1,5 mL. Componentes do tampão de lise terá de ser otimizado para o tipo de célula e as condições de crescimento. Um buffer bom começo seria de 20 mM HEPES-KOH, pH 7,4, 15 mM MgCl 2, 200 mM KCl, 1% Triton X-100 (v / v), 100 mcg / mL cicloheximida, 2 mM DTT e 1 mg / heparina mL. As variáveis-chave são as concentrações de magnésio e cloreto de potássio, bem como a inclusão ou exclusão de detergentes não-iônicos para melhorar a extração do citoesqueleto ou membrana polissomos ligados. Lisar células, passando por 27 gauge agulha da seringa ou duas vezes por golpes de homogeneização 10/05 Dounce usando Tipo B pilão. A seringa ou homogeneizador deve ser pré-refrigerado antes de usar. </li><li> Giram a 14.000 xg por 5 min em microcentrífuga refrigerada. Transferência lisado fresco para o gradiente de sacarose (para células de mamíferos esta é feita em tampão de lise falta Triton X-100 e heparina, mas de outra forma, como descrito em 1.2 e 1.3) ou congelar flash em nitrogênio líquido para uso posterior. </li></ol&gt; 4. Centrifugação de gradientes <ol><li> Descongelar lisados ​​polyribosome no gelo. Determinar a concentração de ácido nucléico na lisado, adicionando a amostra de 10 mL reservados a 1 mL de água e leitura do OD 260 / 280 OD relação com um espectrofotômetro. Rendimento típico de uma cultura de leveduras cultivadas como descrito é de 50 unidades de OD. </li><li> Com uma pipeta com a ponta colocado contra a parede do tubo perto da superfície do gradiente, gentilmente camada de 25 OD 260 unidades do lisado no topo do gradiente. Para um gradiente de 35 mL 500 mL de lisado é normalmente carregado. Lise pode ser usado para garantir a todos os gradientes são carregados com um volume igual. Menor quantidade de material resultam em melhor resolução. Utilizando uma pinça, cuidadosamente inferior os gradientes em baldes de um rotor de caçamba móvel. </li><li> Centrifugar os gradientes de 100.000 xg (23.000 rpm em um 630 Surespin rotor) por 4 horas a 4 ° C. Este protocolo pode ser adaptado para gradientes de menor volume. </li></ol> 5. Recolha de dados e frações <ol><li> Cerca de 30 min antes da conclusão do spin hr 4 anexar a agulha para a piercer tubo Modelo 184. Anexar a caneta para o modelo on-line Isco UA-5 Absorbância / Monitor de fluorescência. Poder sobre a absorbância monitor para permitir uma linha de base estável para ser alcançado antes de amostras. Aquisição eletrônica do perfil polysome pode ser facilmente obtido anexando um DI-148U unidade de aquisição de dados (DATAQ Instruments) para o registrador gráfico. Os perfis podem ser coletados em tempo real e guardado para mais tarde anotação usando o software que acompanha WinDaq. </li><li> Encha a bomba de seringa com 50 ml de Fluorinert usando o fluxo reverso, a definição rápida. Certifique-se que não há bolhas de ar. Ligue a mangueira da seringa para o piercer tubo e brevemente ligar o fluxo de Fluorinert a 3 mL / min para a frente para limpar a mangueira de bolhas de ar. </li><li> Coloque um copo de resíduos ou de uma série de tubos de coleta de frações se sob a mangueira no final da célula de fluxo para coletar amostra de fugir. </li><li> Remova cuidadosamente os tubos polyallomer centrífuga contendo o gradiente de sacarose de rotor de centrífuga e colocá-los no gelo (pré-forma os poços no gelo com um tubo vazio para evitar gradientes de perturbação). </li><li> Para analisar extratos de células, coloque o tubo de gradiente na piercer tubo. Conecte o topo do tubo à saída e seguro. Elevar o estágio inferior para o tubo de centrifugação, para que a agulha perfura o fundo do tubo. Uma vez que a agulha se projetava através do fundo do tubo de centrifugação, comece o fluxo de Fluorinert na direção para frente na 6mL/min. </li><li> Uma vez que o Fluorinert começou fluir para dentro do tubo, monitorar o movimento da agulha no monitor absorbância online. Uma vez que a agulha começa a se mover, girar sobre o movimento gráfico para um ajuste de velocidade de 60. O monitor irá gravar o OD 254 começando com o material livre seguido por subunidade ribossômica 40S detecção e continuando até aos complexos polyribosome. Se você está coletando frações de RNA ou análise de proteínas, tipicamente 0,2 min frações são tomadas (1 mL). Para a análise de RNA, as frações devem ser coletadas diretamente em tubos contendo Trizol (Invitrogen), fenol ou SDS / proteinase K, dependendo do método preferido de extração. </li><li> Quando o fim do perfil polyribosome foi alcançado, desligue o fluxo de Fluorinert para o tubo de centrifugação e parar o movimento gráfico da absorbância / monitor de fluorescência. </li><li> Retrair o Fluorinert do tubo de centrifugação para a seringa, começando o fluxo no sentido inverso a uma taxa rápida tomando cuidado para não chamar a sacarose do tubo para dentro da seringa. Tubo de desapertar a centrífuga do piercer tubo, menor a agulha, e retire o tubo. </li><li> Repita os passos 5,5 por 5,8 para cada gradiente de sacarose. </li><li> Quando todos os gradientes de sacarose foram analisados, recolher o Fluorinert da mangueira para dentro da seringa. Retire cada parte do fraccionamento (piercer tubo e célula de fluxo), incluindo a mangueira de resíduos localizados na parte superior e enxaguar bem com água. </li></ol> 6. Resultados representante <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Figura 1" /> Figura 1. Trace Representante do extrato ribossomo preparados a partir de cepa de levedura BY4741 (mat um his3 Δ Δ 0 1 leu2 met15 Δ 0 Δ ura3 0) na presença de cicloheximida. O extrato foi fracionado ribossomal utilizando um gradiente de sacarose 7 47% e analisados ​​utilizando um aparelho de bomba de seringa conectada a um detector UV. Picos contendo polirribossomos e 80S, 60S e 40S ribossomos são indicados.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

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As informações obtidas a partir do perfil polyribosome pode fornecer informações valiosas sobre o estado de translação da célula. Além disso, o status do conjunto de subunidades ribossômicas se pode ser determinado 2. Por exemplo, a presença de halfmers ou 80 e picos maiores, com um ombro ligeira à direita do perfil indica uma subunidade 40S ligado aguardando subunidade 60S aderir. Realizar o experimento, na ausência de qualquer cicloheximida adicionado ou outros inibidores de alongamento permite a análise da taxa de fugir, o que indica se ou não de alongamento é alterado 3. As frações em si são uma rica fonte de reagentes para a determinação posterior da associação de um mRNA específico ou proteína ribossomal com subpopulações borrando Norte ou Oeste do frações. O grupo total de mRNAs associados ribossomos ativo pode ser determinado através de um microarray associados 4 ou análise de seqüenciamento de profundidade 5. As associações de proteína também pode ser estabilizada pela adição adequada de um cruzamento que liga reagente 6. Polyribosome análise de células de mamíferos Vale destacar também como um protocolo distinto em termos de dificuldades aparentes em estabelecer as condições necessárias para obter polissomos estável. Os sistemas tampão relatados na literatura são muito variáveis ​​7-10, assim pode haver uma exigência para a célula tipo específico de otimização do tampão de lise, ou é igualmente plausível que o sistema de buffer não é tão importante quanto a atenção ansiosos para trabalhar com lisados ​​frescos mantidos em baixas temperaturas. Ao nosso conhecimento uma avaliação sistemática dos parâmetros que afetam a formação polysome mamíferos não tem sido relatada.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

Síntese de proteínas é um processo complexo celular que é regulado em vários níveis. Por exemplo, a tradução global pode ser inibido na fase de iniciação ou a fase de alongamento por uma variedade de estresses celulares, tais como a fome aminoácido ou a retirada do fator de crescimento. Alternativamente, tradução de mRNAs individuais podem ser regulados por Localização mRNA ou a presença de microRNAs cognato. Estudos de síntese de proteína freqüentemente utilizam análise polyribosome para lançar luz sobre os mecanismos de regulação de tradução ou defeitos na síntese de proteínas. Neste ensaio, mRNA / ribossomo complexos são isolados a partir de células eucarióticas. Um gradiente de densidade de sacarose separa mRNAs obrigado a ribossomos vários conhecidos como polirribossomos de mRNAs vinculado a um único ribossomo ou monosome. Fracionamento dos gradientes permite isolar e quantificar as diferentes populações ribossomal e seus mRNAs associados ou proteínas. Diferenças na proporção de polirribossomos para monosomes sob condições definidas podem ser indicativos de defeitos em qualquer de iniciação de tradução ou alongamento / terminação. Exame dos mRNAs presentes nas frações polyribosome podem revelar se a coorte de mRNAs individuais sendo traduzido muda com condições experimentais. Além disso, a montagem ribossomo pode ser monitorada pela análise das pequenas e grandes picos subunidade ribossômica que também são separados pelo gradiente. Neste vídeo, apresentamos um método para a preparação de extratos bruto ribossomal de células de levedura, a separação do extrato por gradiente de sacarose e interpretação dos resultados. Este procedimento é facilmente adaptável para células de mamíferos.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

Este artigo descreve um protocolo para a extração de traduzir os ribossomos das células eucarióticas. Uma vez extraído, ribossomos são separados em monosomes e polirribossomos por fracionamento gradiente de sacarose para permitir diferentes populações ribossomal a ser analisado. Como tal, este método é o padrão ouro para examinar a regulamentação da tradução.

Análise de Perfil eucarióticas Polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.5K visualizações.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. Este artigo descreve um protocolo para a extração de traduzir os ribossomos das células eucarióticas. Uma vez extraído, ribossomos são separados em monosomes e polirribossomos por fracionamento gradiente de sacarose para permitir diferentes populações ribossomal a ser analisado. Como tal, este método é o padrão ouro para examinar a regulamentação da tradução.

Vídeo: Eukaryotic Polyribosome Profile Analysis

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Análise immunoblot

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Biologia

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

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

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 ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Experimento Perfil bioenergética usando células C2C12 mioblastos

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Microscópio óptico e Eletrônica (CLEM) como uma ferramenta para visualizar moléculas microinjetados e seus eucarióticas Sub-celulares Metas


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Estabelecimento de Culturas Microbianas enriquecimento eucarióticas a partir de um quimicamente estratificada Antarctic Lake e Avaliação do Potencial de Fixação de Carbono

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Análise do Perfil Metabólico de embriões Zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Tempo real Análise do Perfil metabólico em<em&gt; Ex Vivo</em&gt; Culturas mouse Intestinal Crypt organóide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

A purificação de todo o genoma de DNA circular Extracromossômica de células eucarióticas

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Protocolo do laboratório para análises genéticas de conteúdo intestino de rDNA usando de macroinvertebrados aquáticos grupo-específicos Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Preparação de organismos procariotas e eucariotas, utilizando secagem química para análise morfológica em microscopia eletrônica (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...