Vanessa Garcia is funded by a Queen Elizabeth II Ontario Graduate Scholarship in Science and Technology and Cory Batenchuk by a Natural Sciences and Engineering Research Council fellowship.

1. Preparación de muestras <ol><li> Obtener muestras que contienen luciferasa que expresa el virus (en el presente documento VSVΔ51-Fluc como ejemplo) para la titulación y la transferencia en placas de 96 pocillos. Alternativamente, realizar infecciones en placas de 96 pocillos de cultivo de tejidos y sobrenadantes de uso directamente. </li><li> Deja dos columnas no tratados para la inclusión de las curvas de calibración. Para experimentos realizados directamente en placas de 96 pocillos, título al final del experimento (40 horas después de la infección) o almacenar a -80 ° C y el título en una fecha posterior. </li></ol> 2 Preparación de células permisivas para la titulación del virus <ol><li> 24 horas antes de la titulación, preparar una suspensión de células Vero a una concentración de 2,5 x 10 5 células / ml en medio Eagle modificado de Dulbecco (DMEM) que contenía 10% de suero fetal bovino (FBS), 30 mM HEPES, y 1% de penicilina / estreptomicina. NOTA: Aunque este protocolo utiliza células Vero, cualquier línea celular permisiva adecuado para la infección por VEV podría abe utiliza. </li><li> Seed 2.5 x 10 4 células (100 l) en sólidas placas de fondo plano de 96 pocillos blancos usando un dispensador de microplacas. <ol><li> Preparar 12 ml de suspensión de células por placa, además de 5 ml para el cebado. </li><li> Clean microplaca dispensador de casete mediante el lavado del tubo con 50 ml de agua estéril. </li><li> Llenar las líneas dispensador de microplacas con suspensión de células y dejar 5 ml de suspensión de células fluyen a través. </li><li> Seleccionar un programa que prescindir de 100 l en cada pocillo de una placa de 96 pocillos de paredes blancas. Dispense, y repetir según sea necesario para las placas adicionales. También sembrar unos pozos en una placa de fondo plano de 96 pocillos claro si las placas de paredes blancas opacas inferior se utilizan para la verificación de la salud celular y la densidad. </li><li> Cuando haya terminado, las células ras de espalda en el recipiente original. Limpiar el casete mediante la ejecución de 50 ml de etanol al 70% seguido de 50 ml de agua estéril caliente a través de la tubería. </li><li> Asegúrese de cassette y tubo son apcuadamente limpiada entre usos. </li></ol></li><li> Se incuban las células durante 24 horas a 37 ° C en un humidificado 5% de CO 2 incubadora. </li></ol> 3. Preparación de la curva estándar Viral <ol><li> Preparar una curva estándar de VSVΔ51-Fluc en DMEM libre de suero tal que la concentración final de unidades formadoras de placa (pfu) por ml después de la transferencia en células Vero es la siguiente: 10 8 pfu / ml, 10 7 pfu / ml, 10 6 pfu / ml, 10 5 pfu / ml, 10 4 pfu / ml, 10 3 pfu / ml, 10 2 pfu / ml, y 10 1 pfu / ml. Preparar 50 l de cada concentración por placa de células Vero, más un 10% adicional. NOTA: título del stock de virus luciferasa tendrá que ser evaluado de una manera clásica 10 con el fin de generar una curva estándar que permita una cuantificación absoluta. De lo contrario, la cuantificación relativa se puede lograr sin información precisa título estableciendo arbitrariamente título viral basaen pasos de dilución. Por ejemplo, la primera dilución de la curva estándar se puede establecer en 10 8 unidades virales y la dilución 1/10 a 10 7 siguiente y así sucesivamente. En este contexto, se debe expresar valores como el cambio veces en comparación con una muestra pre-determinado como la cuantificación absoluta no será exacta. </li></ol> 4. Transferencia de sobrenadantes de ejemplo en células permisivas <ol><li> Compruebe células Vero en placas en la placa de 96 pocillos de fondo claro con bajo un microscopio de luz para confirmar que las monocapas son al menos el 95% de confluencia. </li><li> Transferir 25 l de muestra de sobrenadante sobre células Vero sembradas en placas de paredes blancas. No transfiera sobrenadantes en las 2 columnas designadas para las curvas de calibración. NOTA: Esto se puede hacer de forma simultánea para todos los pozos en una sola placa usando un manipulador de líquidos de 96 canales. </li><li> Uso de una pipeta multicanal de 8 o 12 canales, añadir 25 l de cada dilución de la curva estándar preparada en la Etapa 3 a las células Vero enlas 2 columnas designadas. </li><li> Centrifugar las placas durante 5 minutos a 430 xg a TA. </li><li> Incubar durante 5 horas a 37 ° C en un humidificado 5% de CO 2 incubadora. </li></ol> 5. Evaluación de la viabilidad de la célula <ol><li> NOTA: Cuando a partir de los sobrenadantes obtenidos a partir de experimentos de infección realizados en placas de 96 pocillos claros, la viabilidad de la muestra se puede evaluar antes de la cuantificación con un colorante indicador de viabilidad celular, tales como resazurina. </li><li> Añadir resazurina en una cantidad igual al 10% del volumen en cada pocillo de la placa de 96 pocillos de muestras que contienen virus y células. Incluir celular sólo controla así como el control de sólo los medios de comunicación para determinar los valores de 100% y 0% de viabilidad, respectivamente. </li><li> Después de 2-4 horas (tiempo de incubación variarán dependiendo del tipo celular y de la concentración de polvo del colorante disponible comercialmente o reconstituida), leer y grabar la señal usando un lector de placas de fluorescencia (530-560 de excitación, 590 de emisión). Informe viabili celularTy para una muestra de acuerdo a la fórmula relativa Actividad Metabólica = ((señal de muestra de prueba - señal de control negativo) / (célula única señal de control - señal de control negativo)) x 100% </li></ol> 6. Preparación del sustrato luciferina <ol><li> 30 min antes del final de la incubación de 5 h, preparar la luciferina para obtener una solución / ml 2 mg en solución salina estéril tamponada con fosfato (PBS). Preparar 2,5 ml por placa, más un extra de 2 ml. Proteja la solución de la luz. NOTA: La luciferina también se puede preparar antes en el día y se almacena a 4 ° C hasta su uso. </li></ol> 7. Reading bioluminiscencia <ol><li> Después del período de incubación de 5 h, se añaden 25 l de luciferina a cada pocillo de células Vero en las placas de sólido blanco. Añadir luciferina de forma manual o con un dispensador automático integrado en el luminómetro. NOTA: El uso de un instrumento con sólo un único punto y final lee puede requerir la adición de una luciferasa buf lisis compatiblefer antes de añadir el sustrato luciferina para mejorar la consistencia. </li><li> Leer las placas con los siguientes parámetros: <ol><li> Agitar durante 5 seg. </li><li> Espere 30 seg. </li><li> Leer luminiscencia a un valor apropiado de sensibilidad / exposición fija. Si la opción está disponible en el instrumento, utilice múltiples puntos lee (por ejemplo, 3 x 3 de la matriz) para mejorar la precisión. </li></ol></li><li> Grabar y guardar la intensidad bioluminiscente cuantificado. </li><li> Si se utilizó un dispensador automático para agregar luciferina purgar la luciferina y cebar las líneas con agua estéril tibia. </li></ol> Análisis de datos 8. <ol><li> Para obtener resultados precisos, resolver la regresión no lineal para generar una ecuación de Hill partir de las curvas estándar. Aplicar esta ecuación para las muestras tituladas para calcular unidades de expresión virales. Algunos virus o situaciones pueden producir una curva estándar con una relación lineal; en este caso, para resolver una ecuación lineal. </li></ol>

<ol>
	<li>Grigorov, B., Rabilloud, J., Lawrence, P., Gerlier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+titration+of+measles+and+other+viruses:+optimization+with+determination+of+replication+cycle+length.">Rapid titration of measles and other viruses: optimization with determination of replication cycle length.</a> PLoS One. 6 (e24135), (2011).</li><li>Lizee, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum Gene Ther. 14, 497-507 (2003).</li><li>McSharry, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uses+of+flow+cytometry+in+virology.">Uses of flow cytometry in virology.</a> Clin Microbiol Rev. 7, 576-604 (1994).</li><li>Watcharatanyatip, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispecies+detection+of+antibodies+to+influenza+A+viruses+by+a+double-antigen+sandwich+ELISA.">Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.</a> J Virol Methods. 163, 238-243 (2010).</li><li>Dawson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+Direct+Quantification+of+Viruses+in+Solution+Using+the+ViroCyt+Virus+Counter.">Rapid, Direct Quantification of Viruses in Solution Using the ViroCyt Virus Counter.</a> Journal of Biomolecular Techniques. 23 (S10), (2012).</li><li>Snyder, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+and+RT-PCR:+true+or+false.">AAV and RT-PCR: true or false.</a> Mol Ther. 1, 389-390 (2000).</li><li>Diallo, J. S., Roy, D., Abdelbary, H., De Silva, N., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+infection+of+live+tissue+with+oncolytic+viruses.">Ex vivo infection of live tissue with oncolytic viruses.</a> J Vis Exp. 52, (2011).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells.">Replication and plaque assay of influenza virus in an established line of canine kidney cells.</a> Appl Microbiol. 16, 588-594 (1968).</li><li>Green, M., Loewenstein, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adenoviruses:+propagation,+purification,+quantification,+and+storage.">Human adenoviruses: propagation, purification, quantification, and storage.</a> Curr Protoc Microbiol. 14 (14C 11), (2006).</li><li>Diallo, J. S., Vaha-Koskela, M., Le Boeuf, F., Bell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation,+purification,+and+in+vivo+testing+of+oncolytic+vesicular+stomatitis+virus+strains.">Propagation, purification, and in vivo testing of oncolytic vesicular stomatitis virus strains.</a> Methods Mol Biol. 797, 127-140 (2012).</li><li>Hoenen, T., Groseth, A., Callison, J., Takada, A., Feldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Ebola+virus+expressing+luciferase+allows+for+rapid+and+quantitative+testing+of+antivirals.">A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals.</a> Antiviral Res. 99, 207-213 (2013).</li><li>Diallo, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+pharmacoviral+approach+identifies+novel+oncolytic+virus+sensitizers.">A high-throughput pharmacoviral approach identifies novel oncolytic virus sensitizers.</a> Mol Ther. 18, 1123-1129 (2010).</li></ol>

The authors have nothing to disclose.

El enfoque basado en la luciferasa descrito aquí ofrece un número de ventajas sobre otros métodos existentes incluyendo su facilidad, rapidez, mínima necesidad de equipo, y relativamente bajo costo. Un elemento clave para esto es la evitación de un paso de dilución en serie. Sin embargo, los derivados basados ​​en dilución de serie de este protocolo son ciertamente factible y se usaron recientemente para evaluar los títulos de Ébola luciferase-expresando en una pantalla antiviral de alto rendimiento 11. Mientras inherentemente más lento y más caro, estas adaptaciones pueden proporcionar un mayor rango dinámico para la cuantificación viral cuando es necesario. Además de ser particularmente adecuado para la evaluación de los títulos virales en el contexto de las pantallas de alto rendimiento, nuestro método de cuantificación viral basado en un solo paso de la luciferasa genera estimaciones precisas de viriones infecciosos en el caso de la replicación de los virus. Además, las lecturas de luminiscencia junto con los datos de citotoxicidad del mismo experimento dan una másimagen completa del efecto de las condiciones experimentales en las células diana, que es particularmente útil en el contexto de los virus oncolíticos y pantallas de drogas. El ejemplo ilustrado aquí utiliza un virus de ARN de una sola hebra negativa en replicación; Sin embargo, este protocolo se puede adaptar a un número de replicar y no replicante virus con unos pocos ajustes menores de protocolo. Esto incluye virus de ADN tales como vaccinia, HSV, y AAV (véase la Figura 5). Las muestras infectadas con virus intracelulares, tales como virus de la vacuna, por ejemplo, requieren un paso de liberación de virus antes de la cuantificación (por ejemplo, al menos un ciclo de congelación-descongelación). Cuando se aplica esta técnica a otros virus, es necesario para optimizar el tiempo de incubación de la transferencia del sobrenadante viral o lisado a la lectura de las placas por el luminómetro. Este parámetro dependerá principalmente en el ciclo de replicación del virus en la línea celular permisiva y la fuerza de la promoter conducir la expresión de luciferasa. Esto se hace mejor mediante el uso de la curva estándar completo en la etapa de optimización. Para hacer esto, uno debe infectar la línea celular permisiva apropiado con varios repeticiones de la curva estándar preparada y leer cada repetición en un momento en diferentes puntos después de la transferencia. Idealmente, se elige un punto de tiempo de incubación que conduce a una relación lineal entre log (RLU) y log (título) que abarca el rango de la muestra título esperado. Para VSVΔ51-Fluc, esto es típicamente de 10 4 pfu / ml -10 7 pfu / ml durante un tiempo de incubación de 5 h. Si se espera que los títulos más bajos o más altos a partir de muestras, uno puede simplemente aumentar o reducir el tiempo de incubación, respectivamente. Alternativamente, las muestras pueden ser diluidas a caer dentro de la gama mucho como se hace normalmente para ELISA. Como se mencionó anteriormente, este método es muy adecuado para realizar pantallas de alto rendimiento de medicamentos utilizando bibliotecas de drogas como la mayoría de los pasos se pueden automatizar. Las células se pueden sembrar efficiently usando un dispensador de microplacas automatizado, la biblioteca de fármacos se puede añadir usando un manipulador de líquidos de 96 canales, el virus se puede añadir usando un dispensador de microplacas y placas de leer usando un luminómetro automatizado. En teoría, esto también se puede adaptar a 384 pocillos o formatos más pequeños; Sin embargo, la limitación a este fin es el número de células que pueden sembrarse, menos células dadas conduce a un rango más estrecho en la linealidad de la LOG (RLU) a LOG (título) relación. Finalmente, la evaluación de la viabilidad celular usando la resazurina o otros colorantes metabólicos se puede incorporar fácilmente en el flujo de trabajo, lo que permite la discriminación de compuestos citotóxicos en las pantallas de antivirales o la identificación de compuestos que conducen a la muerte sinérgica en combinación con virus 12. Sin embargo, las limitaciones de este método incluyen el requisito de un virus transgén que expresa luciferasa, que no siempre es posible, y la disponibilidad de una línea celular permisiva suficientemente. Sin embargo, es probable posible adaptarel método para su uso con otros genes informadores (por ejemplo, GFP) proporcionó el método de cuantificación reportero tiene una linealidad adecuado y relación señal a ruido. En general, el método de alto rendimiento descrito puede ser modificado para adaptarse a muchos virus diferentes y adaptarse a diversas aplicaciones.

Classical viral plaque assays continue to be a mainstay in virus research even though they can be notoriously time-consuming and constitute a significant bottleneck to obtaining results from experiments. More rapid indirect virus quantification methods have emerged including quantitative polymerase chain reaction (qPCR), ELISA, and flow cytometry1-4. Recent innovations such as the Virocyt virus counter can directly count viruses using advanced flow sorting technology and through a combination of protein and DNA/RNA dyes5. While all of these methods have undoubtedly quickened the pace of virus research, each method has its advantages and drawbacks. For example, qPCR can allow for quantification of specific viral genome sequence but cannot effectively discriminate infectious from defective virions6. ELISAs can be very specific however require a suitable antibody against the desired target viral protein and can be very expensive. While flow cytometry technology offers many advantages and has improved significantly, throughput and accessibility to highly specialized equipment nonetheless remains a hurdle. Importantly, all of these techniques are not ideally suited for high-throughput screening, for which the ease and time requirement of the virus quantification step is of critical importance.
Here we describe a high-throughput and easily automatable technique to titer viruses that express a Firefly luciferase (Fluc) transgene. This method generates approximate viral titers in a test sample based on luminescence signal reads through the parallel use of a standard curve of known amounts of virus. Samples containing unknown quantities of luciferase-expressing virus are transferred on to a permissive &#8220;plaquing&#8221; cell line in parallel with the standard virus dilution curve and virus-associated luminescence is read after a few hours incubation time. This allows for rapid, quantitative, often same-day generation of results, unlike classic plaque assay protocols which typically require several days of incubation in order to manually count visible plaques7-9. 
The protocol outlines the steps of our titration method using oncolytic Vesicular Stomatitis Virus encoding a Fluc transgene (VSV&#8710;51-Fluc) as an example and provides an overview of 1. Sample preparation 2. The plating of a permissive cell line for virus titration using an automated dispenser 3. The preparation of the viral standard curve 4. The transfer of the sample supernatants onto the permissive cell line using a 96-well manual pipettor 5. The assessment of sample cytotoxicity using a cell viability reagent 6. The preparation of the luciferin substrate 7. Reading of bioluminescence and 8. Data analysis.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Name of Material/ Equipment</td>
			<td style="width:115px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:251px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbeccos&#39; Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Corning</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>NorthBio Inc.</td>
			<td>NBSF-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Phosphate buffered saline</td>
			<td>Corning</td>
			<td style="width:136px;">21-040-CV</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HEPES</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:136px;">BP310-1</td>
			<td style="width:251px;">Prepare a&#160; 1mM solution, pH 7.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alamarBlue&#160;</td>
			<td>AbD Serotec</td>
			<td>BUF012B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin potassium salt</td>
			<td>Biotium</td>
			<td>10101-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">96-well solid white flat bottom polystyrene TC-treated microplates</td>
			<td>Corning</td>
			<td align="right" style="width:136px;">3917</td>
			<td style="width:251px;">384-well plates can also be used for higher throughput</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy Mx</td>
			<td>BioTek</td>
			<td>SMTBL</td>
			<td>Monochromator microplate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Liquidator96</td>
			<td>Mettler Toledo</td>
			<td>LIQ-96-200</td>
			<td>96 tip manual pipetting system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Liquidator96 LTS Tips sterilized with filters</td>
			<td>Mettler Toledo</td>
			<td style="width:136px;">LQR-200F</td>
			<td style="width:251px;">Any sterile filtered tips compatible with pipettors of choice are appropriate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microflo</td>
			<td>BioTek</td>
			<td>111-206-21</td>
			<td>Used to plate cells in a 96 well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroskan Ascent FL</td>
			<td>Thermo Scientific</td>
			<td>5210450</td>
			<td>Microplate fluorometer</td>
		</tr>
	</tbody>
</table>

high-throughput titration of luciferase-expressing recombinant viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

Un resumen del flujo de trabajo que describe el método de alto rendimiento se ilustra en la Figura 1. Figura 2 muestra los resultados de una curva estándar típica ofVSVΔ51-Fluc. De cuatro parámetros análisis de regresión no lineal genera una parcela colina desde la que desconocido pfu de entrada (estimación de la titulación viral) se puede interpolar. Estos títulos estimados se denominan unidades de expresión virales (VEU). Figura 3A muestra Veus y los títulos obtenidos mediante la realización de un ensayo de placa estándar con las mismas muestras 10. Las muestras procedían de un experimento en el que se utilizaron diversos productos químicos para mejorar la replicación y propagación de VSVΔ51-Fluc en 786-0 células. Veus interpolados a partir de la curva estándar debe ser multiplicado por un factor que se basa en la dilución de los sobrenadantes de la muestra que se transfiere a las células Vero (en este caso, el factor de dilución es 5). La correlación lineal entre los títulos y VEU se muestra en la Figura 3B </strong&gt; Con un R 2 de 0,8083 y la puntuación r de Pearson de 0,899 (p &lt;0,0001). En la Figura 4, los datos de citotoxicidad celular típicos obtenidos a partir de un ensayo metabólico se muestra para 786-0 células tratadas con productos químicos antes de la infección con VSVΔ51-Fluc. Finalmente, la Figura 5 muestra curvas típicas estándar obtenidos con diversos virus (virus del herpes simple (HSV), virus vaccinia, y virus adeno-asociado (AAV), todos expresar luciferasa de luciérnaga) y describe tiempos de incubación y los ciclos de congelación-descongelación, según se requiera. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> Figura 1 Flujo de trabajo de titulación de virus de alto rendimiento y ensayo de citotoxicidad usando VSV Δ 51-Fluc. (A) Seed 2,5 x 10 4 células Vero por pocillo (100 l) y se incuba a 37 ° C. (B) 24horas más tarde la transferencia de 25 l de curva estándar en que las células Vero (2 columnas por placa). (C) Transferencia de 25 l de muestras que se tituló a células Vero restantes. Placas de centrífuga y se incuban a 37 ° C. (D) en una cantidad igual al 10% del volumen en el pozo, añadir reactivo resazurina a la placa que contiene las muestras originales. Incubar las placas a 37 ° C. (E) Después de 3 horas, leer y registrar la fluorescencia y evaluar la citotoxicidad. (F) Después de 5 horas, se añaden 25 l de solución de 2 mg / ml de luciferina a cada pocillo de células Vero. Leer la luminiscencia y calcular unidades de expresión viral. <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> Figura 2. esperada curva estándar de VSV Δ 51-Fluc. Expresión de luciferasa era yoasured 5 hr post transferencia sobrenadante en cinco puntos diferentes dentro de un pocillo utilizando un luminómetro y la bioluminiscencia se expresó en unidades relativas de luz (RLU promedio). La media de RLU se representó frente de entrada conocida pfu / ml para resolver la regresión no lineal y generar una ecuación de Hill. El promedio de dos curvas en paralelo y barras de error estándar se muestra (r 2 = 0.9993). <img alt="Figura 3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> Figura 3 Comparación de los títulos de ensayo de placa estándar con los obtenidos por el método de alto rendimiento. (A) Los títulos virales en pfu / ml obtenida por ensayo de placa estándar en células Vero se compararon con los títulos virales calculados (VEU / ml) de las mismas muestras tituladas utilizando el ensayo de luciferasa de alto rendimiento. (B) relación lineal entre VEU / ml y el título obtenerse a través de p estándarensayo laque. Se muestran curva de regresión lineal y el coeficiente de determinación (R 2). <img alt="Figura 4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> Viabilidad de la muestra antes de la transferencia sobrenadante sobre células Vero se determinó mediante la evaluación de la actividad metabólica celular utilizando una solución de resazurina disponible comercialmente Figura 4 Muestra viabilidad.. Valores de fluorescencia primas se normalizaron a la de los pozos no tratados, no infectados. <img alt="Figura 5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> Figura 5. esperado curvas estándar con HSV, vaccinia, y virus de AAV. La expresión de luciferasa se ​​midió en cinco puntos diferentes dentro de un pocillo utilizando un luminómetro y la bioluminiscencia se expresó en rela mediastivas unidades de luz (RLU). (A) curva estándar HSV se añadió a las células Vero plateado 24 horas antes a una densidad de 2,5 x 10 4 células por pocillo (100 l) y la medición de la luciferasa se ​​realizó 17 h después de la transferencia sobrenadante (R 2 = 0,9489, n = 1). Una ecuación de Hill fue generado mediante la resolución de la regresión no lineal. Curva estándar (B) Vaccinia se añadió a las células Vero chapado 24 hr antes una densidad de 2,5 x 10 4 células por pocillo (100 l) y se incubaron durante 2,5 horas a 37 ° C, después de lo cual se tomaron mediciones de luciferasa (R 2 = 0,9892). Una ecuación lineal fue generada por resolver para la regresión lineal. (C) de AAV curva estándar se añadió a las células de carcinoma de pulmón humano (A549) chapado en 24 horas antes a una densidad de 2,5 x 10 4 células por pocillo (100 l) y la medición de la luciferasa se hizo 24 horas después de la infección. Una ecuación de Hill fue generado mediante la resolución de la regresión no lineal. El promediode se muestran cinco curvas en paralelo y barras de error estándar (R 2 = 0.9926).

Watch this Scientific Journal Video about High-throughput Titration of Luciferase-expressing Recombinant Viruses at JoVE.com

High-throughput Titration of Luciferase-expressing Recombinant Viruses

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

De Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

high-throughput-titration-of-luciferase-expressing-recombinant-viruses

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.8K vistas.  Ottawa Hospital Research Institute. This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Video: High-throughput Titration of Luciferase-expressing Recombinant Viruses

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Investigación

Biología

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

De alto rendimiento de sacarificación de ensayo para materiales lignocelulósicos

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

De alto rendimiento de purificación de afinidad de proteínas marcadas con recombinantes

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microinyecciones de Sea Urchin cigotos

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening Expresión cuantitativa y Purificación Aplicada a Recombinante Disulfide ricos en proteínas del veneno Producido en<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput<em&gt; Danio rerio</em&gt; Ensayo gasto energético

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

De alto rendimiento, de múltiples imágenes Cryohistology de tejidos mineralizados

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

De Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

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