Los autores desean agradecer a John Woolford de la base inicial de este método, TGK es apoyado por el NIH GM057483 y AI076245 y la República Popular China se apoya en GM77073.

1. Preparación de los gradientes de sacarosa 7-47% <ol><li> Prepare gradientes de sacarosa un día antes de su uso para permitir que los gradientes de convertirse en permanente. Hacer estéril el 7% y un 47% las soluciones de sacarosa que contiene 50 mM NH 4 Cl, 50 mM Tris-acetato de pH 7,0 y 12 mm de MgCl 2 y se almacenan a 4 ° C 1. </li><li> Para 6 gradientes mezcla del 7% y el 47% de sacarosa soluciones madre para 48 ml de cada una de las siguientes concentraciones de sacarosa. Añadir DTT (1M acciones) a cada solución de sacarosa a una concentración final de 1mM. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> La concentración final </td><td> 7% de sacarosa </td><td> 47% de sacarosa </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> Con el fin de verter los gradientes, adjunte una pipeta Pasteur a una jeringa de 20 ml de sujeción y cierre con Parafilm o usar una aguja larga. 7 ml de sacarosa al 7% a la parte inferior de 1 x 3.5 &quot;tubo de ultracentrífuga polyallomer. A continuación 7 ml de solución de sacarosa al 17% en la solución de un 7% mediante la colocación de la punta de la pipeta Pasteur cerca del fondo del tubo y la pipeta no mayor a 0.3 mL / seg. Repita con 7 ml cada una de 27%, 37% y 47% soluciones de sacarosa. Usted debe observar las líneas claras entre las capas, lo que indica que un mínimo de mezcla se ha producido. tienda gradientes a 4 ° C durante la noche junto con los discos, botellas de y los tubos que se utilizarán para recoger muestras. </li></ol> 2. Preparación del extracto de levadura <ol><li> Crecen 125 ml de cultivo de levadura a una OD 600 de 0,8 a 1,0. Añadir cicloheximida a la cultura a una concentración final de 100 mg / ml y continuar agitación a 30 ° C durante 15 min. </li><li> Mientras tanto, preparar un tampón de lisis que contiene 80 mg / ml de cicloheximida, 200 heparina mg / ml, 0,2% DEPC, y 10 mM Tris-HCl pH 7,5, 0,1 M NaCl, 30 mM MgCl 2. Mantenga todo en hielo a partir de ahora. </li><li> Vierta la cultura en una botella de 250 ml de centrífuga y se llenan de hielo. Centrifugar a 8000 xg (7.250 rpm en un rotor de SLA-1500) durante 5 min a 4 ° C. Lavar el sedimento una vez con 10 ml de solución amortiguadora de lisis fría y la transferencia de tubos de 15 ml de polipropileno. Centrifugar a 5900 xg (~ 6000 rpm en un rotor de SLA-600TC) durante 3 min a 4 ° C. Resuspender el precipitado en 0,5 ml de tampón de lisis, traslado al tubo de 1,5 ml y añadir 0,5 ml de cuentas refrigerados, horno de vidrio lavado con ácido. Vórtice de las células durante cuatro intervalos de 30 segundos, enfriar el tubo en hielo durante 30 segundos entre cada intervalo. Añadir otro 0,5 ml de solución amortiguadora de lisis en el tubo de 1,5 ml. Centrifugar a máxima velocidad en una microcentrífuga (14.000 rpm en una microcentrífuga Eppendorf 5417R) por 10 min a 4 ° C. Transferir el sobrenadante a un nuevo tubo de microcentrífuga de 1,5 ml. Retire 10 l de un tubo separado para determinar la DO 260 / DO 280 ratio (véase el paso 3.1). Extractos de almacenar a -80 ° C. </li></ol> 3. Preparación de extractos de células de mamíferos <ol><li> Para células adherentes, crecer en un plato de 100 mm hasta la confluencia ~ 70%. Usted necesitará un plato de 100 mm por cada 11 ml de gradiente (rendimiento típico es de ~ 20 OD 260 unidades). Escala de la cantidad lineal para los tamaños de gradiente de mayor o menor. De las células no adherentes, de aproximadamente 1 x 10 7 células son necesarias por gradiente de 11 ml. </li><li> Antes de la lisis, añadir cicloheximida a 100 mg / ml. Se incuba a 37 ° C durante 15 min. Transferencia de las placas de hielo y lavar las células dos veces en PBS enfriado con hielo. Todos los pasos de futuro debe llevarse a cabo a 0-4 º C. </li><li> Eliminar todos los rastros de PBS por aspiración (que las placas de drenaje en una cama inclinada de hielo) y añadir una solución amortiguadora de lisis ml, raspar y la transferencia de pre-enfriado tubo de 1,5 ml. Componentes de tampón de lisis tendrá que ser optimizado para el tipo de célula y las condiciones de crecimiento. Un buffer de partida sería de 20 mM HEPES-KOH, pH 7,4, 15 mM MgCl 2, 200 mM KCl, 1% Triton X-100 (v / v), 100 mg / ml de cicloheximida, 2 mM DTT, y 1 mg / ml de heparina. Las variables clave son las concentraciones de magnesio y cloruro de potasio, así como la inclusión o exclusión de los detergentes no iónicos para mejorar la extracción de polisomas obligado citoesqueleto o de la membrana. Lisis de las células, pasando al 27 de aguja de la jeringa de calibre o dos veces por golpes 10.5 de la homogeneización Dounce con el Tipo B maja. La jeringa o un homogeneizador debe ser pre-enfriado antes de su uso. </li><li> Giran a 14.000 xg durante 5 minutos en microcentrífuga refrigerada. Transferencia de lisado fresco al gradiente de sacarosa (para células de mamíferos esto se hace en tampón de lisis que carecen de Triton X-100 y la heparina, pero de lo contrario como se describe en 1.2 y 1.3) o congelar flash en nitrógeno líquido para su uso posterior. </li></ol&gt; 4. Centrifugación de gradientes <ol><li> Descongele los lisados ​​polyribosome en el hielo. Determinar la concentración de ácido nucleico en el lisado mediante la adición de la muestra de 10 l reservados a 1 ml de agua y la lectura de la DO 260 / DO 280 relación con un espectrofotómetro. Rendimiento típico de un cultivo de levadura de cerveza cultivada como se describe es de 50 unidades de DO. </li><li> Con una pipeta con la punta colocada contra la pared del tubo cerca de la superficie de la pendiente, suave capa de 25 OD 260 unidades del lisado en la parte superior de la pendiente. Para un gradiente de 35 ml 500 l de lisado se carga típicamente. Tampón de lisis se puede utilizar para asegurar que todos los gradientes están cargados con un volumen igual. Una cantidad menor de material de mejor rendimiento de resolución. Con unas pinzas, cuidadosamente baje los gradientes en los cubos de un rotor basculante. </li><li> Centrifugar los gradientes a 100.000 xg (23.000 rpm en un rotor Surespin 630) por 4 horas a 4 ° C. Este protocolo puede ser adaptado para los gradientes de menor volumen. </li></ol> 5. La recopilación de datos y las fracciones <ol><li> Aproximadamente 30 minutos antes de la finalización de la separación de 4 horas coloque la aguja de la perforación del tubo Modelo 184. Coloque el lápiz a la línea de modelo Isco UA-5 Absorbancia / Monitor de fluorescencia. Encienda el monitor de absorción para permitir que una línea de base estable para ser alcanzado antes de analizar las muestras. Electrónicos para la adquisición del perfil de polisomas se puede obtener fácilmente por colocar un dispositivo de DI-148U de adquisición de datos (DATAQ Instruments) para el registro gráfico. Perfiles se puede recoger en tiempo real y se guarda para su posterior anotación usando el software que acompaña WinDaq. </li><li> Llene la bomba de jeringa con 50 ml de Fluorinert utilizando el flujo inverso, de fraguado rápido. Asegúrese de que no hay burbujas de aire. Conecte la manguera de la jeringa en el tubo de perforación y brevemente a su vez en el flujo de Fluorinert a 3 ml / min en dirección hacia adelante para despejar el tubo de burbujas de aire. </li><li> Coloque un vaso de residuos o una serie de tubos de recogida de las fracciones, si debajo de la manguera en el extremo de la celda de flujo para recoger la muestra corriendo. </li><li> Retire con cuidado los tubos de centrifugación polyallomer que contiene el gradiente de sacarosa del rotor de la centrífuga y colocarlos en hielo (pre-forma de los pozos en el hielo con un tubo de vacío para evitar gradientes inquietante). </li><li> Para el análisis de extractos celulares, se coloca el tubo de gradiente en la perforación del tubo. Conecte la parte superior del tubo a la toma y seguro. Levante la etapa final para el tubo de centrífuga de modo que la aguja perfora el fondo del tubo. Una vez que la aguja ha sobresalido a través de la parte inferior del tubo de centrífuga, iniciar el flujo de Fluorinert en la dirección de avance en 6mL/min. </li><li> Una vez que el Fluorinert ha comenzado a fluir en el tubo, vigilar el movimiento de la aguja en el monitor de la absorbancia en línea. Una vez que la aguja comienza a moverse, a su vez en el movimiento gráfico a un ajuste de velocidad de 60. El monitor medirá el diámetro exterior 254 a partir del material libre seguido por la detección de la subunidad ribosómica 40S y continuando a través de complejos polyribosome. Si usted está recogiendo fracciones de ARN o el análisis de proteínas, por lo general 0,2 min se toman fracciones (1 ml). Para el análisis de ARN, las fracciones se deben recoger directamente en tubos con Trizol (Invitrogen), fenol o K SDS / proteinasa, dependiendo del método preferido de extracción. </li><li> Cuando el extremo del perfil polyribosome se ha alcanzado, a su vez el flujo de Fluorinert al tubo de centrífuga y detener el movimiento de la tabla de absorbancia / monitor de fluorescencia. </li><li> Retraer el Fluorinert desde el tubo de centrífuga con la jeringa de inicio del flujo en la dirección contraria a gran velocidad teniendo cuidado de no extraer la sacarosa de la sonda en la jeringa. Desenroscar el tubo de centrífuga de la perforación del tubo, bajar la aguja, y retirar el tubo. </li><li> Repita los pasos 5.5 a 5.8 por cada gradiente de sacarosa. </li><li> Cuando todos los gradientes de sacarosa se han analizado, retraer el Fluorinert de la manguera en la jeringa. Retire cada parte del fraccionador (perforación del tubo y la celda de flujo), incluyendo la manguera de residuos situado en la parte superior y enjuague bien con agua. </li></ol> 6. Resultados representante <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Figura 1" /> Figura 1. Rastro Representante de extracto de ribosoma preparada con la cepa de levadura BY4741 (una estera his3 Δ Δ 0 1 leu2 met15 Δ 0 ura3 Δ 0) en presencia de cicloheximida. El extracto fue fraccionado ribosomal utilizando un gradiente de sacarosa 7 47% y se analizaron mediante un aparato de bomba de jeringa conectada a un detector de UV. Picos que contienen polirribosomas y 80S, 60S y 40S ribosomas se indican.

<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>

La información obtenida a partir del perfil polyribosome puede proporcionar información valiosa sobre el estado de traslación de la célula. Además, el estado de la asamblea de las subunidades ribosómicas se puede determinar 2. Por ejemplo, la presencia de halfmers y 80 años y mayores picos con un hombro ligeramente a la derecha en el perfil indica una subunidad 40S unido a la espera de unirse subunidad 60S. Realizar el experimento en la ausencia de cicloheximida o inhibidores añadidos otros de elongación permite el análisis de la tasa de escurrimiento, que indica si la elongación se altera 3. Las mismas fracciones son una rica fuente de reactivos para la determinación posterior de la asociación de un ARNm o proteína ribosomal con las subpoblaciones por transferencia de Northern o Western de las fracciones. El número total de mRNAs asociados con los ribosomas activos puede ser determinada a través de un asociado de microarrays 4 o profundo análisis de secuenciación 5. Las asociaciones de proteínas también puede ser estabilizada por la adición apropiada de un entrecruzamiento reactivo 6. Polyribosome análisis de las células de la pena mencionar también como un protocolo distinto en términos de las aparentes dificultades en el establecimiento de las condiciones necesarias para obtener polisomas estable. Los sistemas de amortiguación en la literatura son muy variables 70 a 10, por lo tanto no puede ser un requisito para la optimización del tipo de células específicas de la solución amortiguadora de lisis, o también es posible que el sistema de amortiguación no es tan importante como la atención de interesados ​​en trabajar con lisados ​​frescos se mantienen a temperaturas bajas. A nuestro entender una evaluación sistemática de los parámetros que afectan la formación de polisomas mamíferos no se ha informado.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<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

La síntesis de proteínas es un proceso complejo celular que está regulada en muchos niveles. Por ejemplo, la traducción global puede ser inhibido en la fase de iniciación o de la fase de alargamiento por una variedad de estreses celulares, tales como aminoácidos hambre o la retirada del factor de crecimiento. Por otra parte, la traducción de ARNm individuales pueden ser reguladas por la localización del ARNm o la presencia de micro ARN afines. Los estudios sobre la síntesis de proteínas a menudo utilizan el análisis polyribosome para arrojar luz sobre los mecanismos de regulación de la traducción o defectos en la síntesis de proteínas. En este ensayo, el ARNm / ribosoma complejos están aislados de las células eucariotas. Un gradiente de densidad de sacarosa separa ARNm unido a los ribosomas múltiple conocido como polirribosomas de ARNm unido a un ribosoma único o monosome. El fraccionamiento de los gradientes permite el aislamiento y la cuantificación de las diferentes poblaciones ribosomal y sus asociados ARNm o proteínas. Las diferencias en la proporción de polirribosomas a monosomes en unas condiciones determinadas puede ser indicativo de los defectos de iniciación de la traducción sea o elongación / terminación. El examen de los ARNm presentes en las fracciones polyribosome puede revelar si la cohorte de ARNm individuales se traducen los cambios en las condiciones experimentales. Además, el montaje de los ribosomas se puede controlar mediante el análisis de los picos de la subunidad ribosomal pequeñas y grandes que también se separan por el gradiente. En este video, se presenta un método para la preparación de extractos crudos de células de levadura ribosomal, la separación del extracto por gradiente de sacarosa y la interpretación de los resultados. Este procedimiento es fácilmente adaptable a las células de mamíferos.

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

Eukaryotic Polyribosome Profile Analysis

En este artículo se describe un protocolo para la extracción de la traducción de los ribosomas de las células eucariotas. Una vez extraído, ribosomas están separados en monosomes y polirribosomas por fraccionamiento en gradiente de sacarosa para permitir que las diferentes poblaciones de ribosomas para ser analizados. Por lo tanto, este método es el estándar de oro para el examen de la regulación de la traducción.

Análisis eucariotas perfil 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

52.4K vistas.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. En este artículo se describe un protocolo para la extracción de la traducción de los ribosomas de las células eucariotas. Una vez extraído, ribosomas están separados en monosomes y polirribosomas por fraccionamiento en gradiente de sacarosa para permitir que las diferentes poblaciones de ribosomas para ser analizados. Por lo tanto, este método es el estándar de oro para el examen de la regulación de la traducción.

Video: Eukaryotic Polyribosome Profile Analysis

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 neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

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

Investigación

Biología

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 bioenergético utilizando 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.

Luz correlativa y Microscopía Electrónica (CLEM) como una herramienta para visualizar moléculas microinyección y sus objetivos eucariotas subcelulares


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.

Establecimiento de cultivos de enriquecimiento microbianos eucariotas a partir de un estratificado químicamente lago de la Antártida y evaluación del potencial de carbono de fijación

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.

Análisis del perfil metabólico de embriones de pez cebra

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.

Bienes Análisis Tiempo de perfil metabólico en<em&gt; Ex Vivo</em&gt; Culturas ratón intestinal Cripta organoides

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.

La purificación de todo el genoma de ADN circular Extracromosómica a partir de células eucariotas

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 de laboratorio para análisis genéticos de contenido intestinal de macroinvertebrados acuáticos con grupo-específica rDNA cartillas

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.

Preparación de los organismos procarióticos y eucarióticos con secado químico para análisis morfológico en microscopía electrónica de barrido (SEM)

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

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

Técnicas de renderizado de imágenes en tomografía computarizada postmortem: evaluación de la salud biológica y perfil en cetáceos trenzados

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...