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.

Preparação 1 Amostra <ol><li> Obtenção de amostras contendo vírus que expressam a luciferase (aqui VSVΔ51-Fluc como exemplo) para a titulação e a transferência para placas de 96 poços. Alternativamente, realizar infecções em placas de 96 poços de cultura de tecidos e os sobrenadantes usar diretamente. </li><li> Deixe duas colunas não tratadas para a inclusão de curvas padrão. Por experiências feitas directamente nas placas de 96 poços, de título no final da experiência (40 horas pós-infecção) ou armazenar a -80 ° C e titulação em data posterior. </li></ol> 2: Preparação de células permissivas para o vírus da Titulação <ol><li> 24 h antes da titulação, preparar uma suspensão de células Vero, a uma concentração de 2,5 x 10 5 células / ml em meio de Eagle modificado por Dulbecco (DMEM) contendo 10% de soro fetal bovino (FBS), HEPES 30 mM, e 1% de penicilina / estreptomicina. NOTA: Apesar de este protocolo utiliza células Vero, qualquer linha celular permissiva adequado para uma infecção VSV poderia be usado. </li><li> Semente de 2,5 x 10 4 células (100 ul) em placas de fundo plano de 96 poços sólidos brancos, utilizando um dispensador de microplacas. <ol><li> Preparar 12 ml de suspensão de células por placa de mais 5 ml de preparação. </li><li> Limpo microplaca cassete dispensador, através de lavagem com o tubo de 50 ml de água estéril. </li><li> Encha as linhas de microplacas dispensador com suspensão de células e deixou 5 ml de suspensão de células fluir. </li><li> Selecione um programa que irá dispensar 100 mL em cada poço de uma placa de 96 poços de paredes brancas. Dispensar, e repetir se necessário para placas adicionais. Sementes também alguns poços numa placa de 96 poços de fundo plano transparente se placas de paredes brancas opacas de fundo são usados ​​para verificação da saúde das células e densidade. </li><li> Quando terminar, lave as células de volta para o recipiente original. Limpar o cartucho através da execução de 50 ml de etanol a 70% seguido por 50 ml de água esterilizada quente através do tubo. </li><li> Certifique-cassete e tubos são apdamente limpo entre os usos. </li></ol></li><li> Incubar as células durante 24 horas a 37 ° C numa atmosfera humidificada com 5% de CO 2 incubadora. </li></ol> 3 Preparação da curva padrão viral <ol><li> Prepara-se uma curva padrão de VSVΔ51-Fluc em DMEM isento de soro, tal que a concentração final de unidades formadoras de placas (pfu) por ml depois de se transferir para as células Vero, é como se segue: 10 8 pfu / ml, de 10 7 pfu / ml, 10 6 pfu / ml, de 10 5 pfu / mL, 10 4 ufp / mL, 10 3 ufp / mL, 2 10 pfu / ml, e 10 1 ufc / ml. Prepare 50 mL de cada concentração por placa de células Vero, acrescido de um adicional de 10%. NOTA: Titer do estoque de vírus luciferase terá de ser avaliado de uma forma clássica de 10, a fim de gerar uma curva padrão que permita a quantificação absoluta. Caso contrário, a quantificação relativa pode ser obtida sem informação precisa titulação ajustando arbitrariamente título viral baseadoem passos de diluição. Por exemplo, a primeira diluição da curva padrão pode ser definido como 10 8 unidades virais e a diluição 1/10 a 10 seguintes e assim por diante 7. Neste contexto, deve-se expressar valores como um fold-change em comparação com uma amostra pré-determinada como a quantificação absoluta não será preciso. </li></ol> 4. Transferência de sobrenadantes de exemplo para células permissivas <ol><li> Verificar as células Vero plaqueadas no fundo claro-placa de 96 poços sob um microscópio de luz para confirmar que as monocamadas são pelo menos 95% confluentes. </li><li> Transferir 25 mL de amostra de sobrenadante sobre as células Vero semeados em placas de paredes brancas. Não transferir sobrenadantes nas duas colunas designadas por curvas padrão. NOTA: Este pode ser realizado simultaneamente em todos os poços de uma única chapa através de um manipulador de líquidos de 96 canais. </li><li> Usando uma pipeta de multi-canal 8 ou 12 canais, adicionar 25 ul de cada diluição da curva padrão preparada no Passo 3 para as células Veroas duas colunas designadas. </li><li> Centrifugar as placas durante 5 min a 430 xg à temperatura ambiente. </li><li> Incubar durante 5 horas a 37 ° C numa atmosfera humidificada com 5% de CO 2 incubadora. </li></ol> 5. avaliação da viabilidade celular <ol><li> NOTA: Ao iniciar a partir de sobrenadantes obtidos a partir de experiências de infecção Realizados em claras placas de 96 poços, a viabilidade da amostra pode ser avaliada antes da quantificação com um corante indicador de viabilidade celular, tais como resazurina. </li><li> Adicionar resazurina em uma quantidade igual a 10% do volume, em cada poço da placa de 96 poços de amostras que contêm vírus e as células. Incluir celular só controla bem como o controle apenas pela mídia para determinar os valores de 100% e 0% de viabilidade, respectivamente. </li><li> Depois de 2-4 horas (tempo de incubação pode variar, dependendo do tipo de célula e da concentração de pó de corante comercialmente disponível ou reconstituído), ler e gravar o sinal utilizando um leitor de fluorescência de placas (530-560 excitação, 590 de emissão). Relatório viabili celularty para uma amostra de acordo com a fórmula de Atividade = ((sinal da amostra de teste - sinal de controle negativo) / (Cell só sinal de controle - sinal de controle negativo)) Relativa Metabólica x 100% </li></ol> 6 Preparação do substrato luciferina <ol><li> 30 minutos antes do final do período de incubação de 5 horas, a luciferina preparar para se obter uma solução / ml 2 mg em tampão fosfato salino estéril (PBS). Prepare 2,5 ml por placa, mais um extra de 2 ml. Proteger a solução da luz. NOTA: A luciferina pode também ser preparado no início do dia e armazenadas a 4 ° C até à sua utilização. </li></ol> 7 Leitura Bioluminescência <ol><li> Após o período de incubação de 5 h, adicionar 25 ul de luciferina para cada poço de células Vero em placas de sólidos brancos. Adicionar luciferina manualmente ou com um distribuidor automático integrados no luminómetro. NOTA: O uso de um instrumento com apenas um único ponto e no final lê pode exigir a adição de um buf lise compatível luciferasefer antes da adição do substrato luciferina para melhorar a consistência. </li><li> Ler as placas com os seguintes parâmetros: <ol><li> Agitar durante 5 seg. </li><li> Espere 30 segundos. </li><li> Leia luminescência em um valor de sensibilidade / exposição fixa apropriado. Se a opção estiver disponível no instrumento, use multi-ponto lê (por exemplo, 3 x 3 matriz) para melhorar a precisão. </li></ol></li><li> Gravar e salvar a intensidade bioluminescente quantificado. </li><li> Se um distribuidor automático foi usado para adicionar luciferina purgar a luciferina e injetar as linhas com água esterilizada morna. </li></ol> Análise de Dados 8. <ol><li> Para resultados precisos, resolver a regressão não-linear para gerar uma equação de Hill a partir das curvas padrão. Aplicar esta equação para as amostras tituladas para calcular unidades de expressão virais. Alguns vírus ou situações podem produzir uma curva padrão com uma relação linear; neste caso, para resolver uma equação linear. </li></ol>

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	<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.

A abordagem aqui descrita luciferase oferece um número de vantagens sobre os outros métodos existentes, incluindo a sua facilidade, rapidez, um equipamento mínimo necessário, e de custo relativamente baixo. Um contribuidor chave para isso é a prevenção de uma etapa de diluição serial. No entanto, os derivados à base de diluição em série deste protocolo são certamente viável e recentemente foram utilizados para avaliar expressando luciferase títulos de Ebola em uma tela antiviral de alto rendimento 11. Enquanto inerentemente mais demorado e mais caro, essas adaptações podem proporcionar uma maior gama dinâmica para quantificação viral quando necessário. Além de ser particularmente adequado para avaliar os títulos virais no contexto de telas de alto rendimento, o nosso método de quantificação viral a partir de uma etapa de luciferase gera estimativas precisas dos viriões infecciosos, no caso de se replicar os vírus. Além disso, as leituras de luminescência ao longo de citotoxicidade com dados a partir do mesmo experimento dar um maisimagem completa do efeito das condições experimentais em células alvo, o que é especialmente útil no contexto de vírus oncolí ticos e telas de drogas. O exemplo mostrado aqui utiliza um vírus RNA fita única replicando negativo; No entanto, este protocolo pode ser adaptado a um número de replicação e não replicação de vírus com alguns ajustamentos menores do protocolo. Isto inclui os vírus de ADN tais como Vaccinia, o HSV, e de AAV (ver Figura 5). As amostras infectadas com os vírus intracelulares, tais como vírus vaccinia, por exemplo, requerem um passo de libertação antes da quantificação do vírus (por exemplo, pelo menos um ciclo de congelação-descongelação). Quando se aplica esta técnica para outros vírus, o que é necessário para optimizar o tempo de incubação da transferência do sobrenadante viral ou lisado para a leitura das placas pelo luminómetro. Este parâmetro depende principalmente no ciclo de replicação do vírus na linha celular permissiva e a força do promoter dirigir a expressão da luciferase. Este é o melhor feito usando a curva padrão completo na etapa de otimização. Para fazer isso, é necessário infectar a linha celular permissiva apropriado com várias repetições da curva padrão preparada e ler cada replicar num momento diferente de pontos de pós-transferência. Idealmente, um ponto de tempo de incubação é escolhido de que conduz a uma relação linear entre LOG (RLU) e LOG (título) que abrange o intervalo de amostra título esperado. Para VSVΔ51-Fluc, este é tipicamente a partir de 10 4 pfu / ml -10 7 pfu / ml para um tempo de incubação de 5 h. Se os títulos mais baixos ou mais elevados são esperados a partir de amostras, pode-se simplesmente aumentar ou reduzir o tempo de incubação, respectivamente. Como alternativa, as amostras podem ser diluídas para cair dentro da gama muito como é feito tipicamente por ELISA. Como mencionado acima, este método é também adequado para realizar telas de alto rendimento de drogas usando bibliotecas de drogas como a maior parte dos passos pode ser automatizado. As células podem ser plaqueadas efficiently utilizando um dispensador automático de microplacas, a biblioteca de fármacos pode ser adicionado utilizando um manipulador de líquidos de 96 canais, o vírus pode ser adicionado utilizando um dispensador de microplacas e placas lida utilizando um luminómetro automatizado. Em teoria, este também pode ser adaptado para 384 poços ou formatos mais pequenos; no entanto, a limitação para este fim é o número de células que podem ser banhados, dadas menos células leva a uma faixa mais estreita na linearidade do LOG (RLU) a relação log (Titer). Finalmente, avaliação da viabilidade celular utilizando resazurina ou outros corantes metabólicos podem ser facilmente incorporados no fluxo de trabalho, permitindo a discriminação de compostos citotóxicos em telas antivirais ou identificação de compostos que levam à morte sinérgica em combinação com o vírus 12. No entanto, as limitações deste método incluem a necessidade de um vírus que expressa transgene da luciferase, o que nem sempre é possível, e a disponibilidade de uma linha celular suficientemente permissiva. No entanto, é provável possível adaptaro método para ser utilizado com outros genes repórter (por exemplo, GFP), desde que o método de quantificação repórter tem uma linearidade adequada e a relação sinal-ruído. No geral, o método de alto rendimento descrito pode ser modificado para atender muitos vírus diferentes e adaptadas às diversas aplicações.

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.

Um resumo do fluxo que descreve o método de alto rendimento é ilustrada na Figura 1. Figura 2 mostra os resultados de uma curva padrão típica ofVSVΔ51-Fluc. Quatro parâmetros de análise de regressão não-linear gerada uma trama monte a partir do qual desconhecido pfu de entrada (estimativa do título viral) podem ser interpolados. Estes títulos são estimados denominado unidades de expressão viral (VEU). Figura 3A mostra Veus e os títulos obtidos através da realização de um ensaio de placas padrão com as mesmas amostras 10. As amostras provenientes de um experimento em que foram utilizados vários produtos químicos para aumentar a replicação e disseminação de VSVΔ51-Fluc em 786-0 células. Veus interpolado a partir da curva padrão tem de ser multiplicado por um factor que é baseado na diluição de sobrenadantes das amostras serem transferidos para células Vero (no presente caso, o factor de diluição é 5). A correlação linear entre os títulos de VEU e é mostrado na Figura 3B </strong&gt; Com um R 2 de 0,8083 e pontuação r de Pearson de 0,899 (p &lt;0,0001). Na Figura 4, os dados típicos de citotoxicidade de células obtidas a partir de um ensaio metabólico é mostrado para 786-0 células tratadas com produtos químicos antes da infecção com VSVΔ51-Fluc. Finalmente, a Figura 5 mostra curvas típicas padrão obtidas com vários vírus (vírus herpes simplex (HSV), vírus vaccinia e vírus adeno-associado (AAV), todos expressando a luciferase do pirilampo) e descreve os tempos de incubação e dos ciclos de congelamento e descongelamento, tal como requerido. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> Figura 1 Fluxo de trabalho de titulação de vírus de alto rendimento e ensaio de citotoxicidade utilizando VSV Δ 51 Fluc. (A) 2,5 x 10 Semente 4 células Vero por poço (100 ul) e incubar a 37 ° C. (B) 24hr depois de transferência 25 ul de curva padrão sobre a células Vero (2 colunas por placa). (C) Transferir 25 uL de amostras para ser titulado em células Vero às restantes. Centrifugar as placas e incuba-se a 37 ° C. (D) numa quantidade igual a 10% do volume da cavidade, adicionar o reagente de resazurina a placa contendo as amostras originais. Incubar as placas a 37 ° C (E). Depois de 3 horas, ler e registar a fluorescência e avaliar a citotoxicidade. (F) Depois de 5 h, adicionar 25 ul de uma solução 2 mg / ml de luciferina para cada poço de células Vero. Leia luminescência e calcular unidades de expressão virais. <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> Figura 2. esperado curva padrão de VSV Δ 51-Fluc. Expressão da luciferase era measured 5 hr transferência pós sobrenadante em cinco pontos diferentes dentro de um poço usando uma luminometer e bioluminescência foi expresso em médias unidades relativas de luz (RLU). A média de RLU foi representada graficamente contra a entrada conhecida pfu / ml para resolver a regressão não-linear e gerar uma equação de Hill. A média de duas réplicas de curvas e as barras de erro padrão são apresentados (r2 = 0,9993). <img alt="Figura 3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> Figura 3 Comparação de títulos de ensaio de placa padrão com os obtidos pelo método de alto rendimento. (A) Os títulos virais em pfu / ml obtido por ensaio de placas em células Vero padrão foram comparados com os títulos virais calculadas (VEU / ml) a partir das mesmas amostras tituladas utilizando o ensaio de elevado débito da luciferase. (B) a relação linear entre VEU / ml e através de titulação padrão de pensaio laque. Curva de regressão linear e coeficiente de determinação (R 2) são mostrados. <img alt="Figura 4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> Figura 4 Amostra viabilidade. Viabilidade amostra antes da transferência de sobrenadante de células Vero foi determinada avaliando a actividade metabólica celular, utilizando uma solução resazurina disponível comercialmente. Os valores de fluorescência em bruto foram normalizados para a de não tratadas não infectadas, poços. <img alt="Figura 5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> Figura 5 Esperado curvas padrão com HSV, vaccinia e vírus AAV. Expressão da luciferase foi medida em cinco pontos diferentes dentro de um poço, utilizando um luminómetro e bioluminescência foi expressa em rela médiosunidades de luz tiva (RLU). (A) HSV curva padrão foi adicionado em células Vero plaqueadas 24 horas antes a uma densidade de 2,5 x 10 4 culas por po (100 ul) e medição da luciferase foi feita 17 horas pós transferência sobrenadante (R2 = 0,9489, n = 1). Uma equação de Hill foi gerada pela solução da regressão não-linear. (B) Vaccinia curva padrão foi adicionado em células Vero chapeado 24 h antes de uma densidade de 2,5 x 10 4 culas por po (100 ul) e incubou-se durante 2,5 horas a 37 ° C, após o que foram tomadas medidas de luciferase (R2 = 0,9892). Uma equação linear foi gerado por resolver para a regressão linear. (C) de AAV curva padrão foi adicionado em células de carcinoma de pulmão humano (A549) chapeado 24 horas antes a uma densidade de 2,5 x 10 4 culas por po (100 ul) e medição da luciferase foi feito 24 horas após a infecção. Uma equação de Hill foi gerada pela solução da regressão não-linear. A médiade cinco curvas em duplicado eo barras de erro padrão são mostrados (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 alta capacidade de Titulação de luciferase expressando recombinantes Vírus

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

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JoVE Journal

Biology

12.8K visualizações.  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.

Vídeo: 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.

Alto rendimento Yeast Tela superexpressão Plasmid

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

Biologia

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.

Alto rendimento Assay Sacarificação para materiais 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 rendimento de purificação de afinidade com tag Proteínas 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 microinjeções de ouriço do mar zigotos

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 expressão quantitativa e Purificação Aplicada à recombinante Dissulfeto ricos em proteínas veneno produzido em<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.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

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.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

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.

Identificação de pares Kinase-substrato utilizando 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 ...

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; Energia Despesas 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 ...

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 rendimento, Multi-Imagem Cryohistology de mineralizadas tecidos

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