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.

Preparazione del campione 1 <ol><li> Ottenere campioni contenenti-luciferasi che esprimono virus (qui VSVΔ51-Fluc come esempio) per la titolazione e il trasferimento in piastre da 96 pozzetti. In alternativa, eseguire infezioni in 96 pozzetti piastre di coltura di tessuti e uso surnatanti direttamente. </li><li> Lascia due colonne non trattati per l&#39;inserimento di curve standard. Per gli esperimenti fatti direttamente in piastre a 96 pozzetti, titolo alla fine dell&#39;esperimento (40 ore post-infezione) o conservare a -80 ° C e titolo in una data successiva. </li></ol> 2 Preparazione di cellule permissive per Virus Titolazione <ol><li> 24 ore prima della titolazione, preparare una sospensione di cellule Vero ad una concentrazione di 2,5 x 10 5 cellule / ml in mezzo di Eagle modificato di Dulbecco (DMEM) contenente siero bovino fetale al 10% (FBS), HEPES 30 mM e 1% di penicillina / streptomicina. NOTA: Anche se questo protocollo utilizza cellule Vero, ogni linea cellulare permissiva adatto per l&#39;infezione da VSV potrebbe be utilizzato. </li><li> Seed 2,5 x 10 4 cellule (100 microlitri) in piastre a fondo piatto solido 96 pozzetti bianchi con un dispenser di micropiastre. <ol><li> Preparare 12 ml di sospensione cellulare per piastra più 5 ml per adescamento. </li><li> Pulire micropiastre cassette dispenser dal lavaggio del tubo con 50 ml di acqua sterile. </li><li> Riempire le linee dispenser micropiastra con sospensione cellulare e lasciare 5 ml di sospensione cellulare fluire attraverso. </li><li> Selezionare un programma che dispensare 100 ml in ciascun pozzetto di una piastra a 96 pozzetti bianco-murato. Dispensare, e ripetere se necessario per piastre supplementari. Sementi anche alcuni pozzi in una chiara piatto fondo piatto da 96 pozzetti, se si utilizzano le piastre bianche pareti opache-fondo per la verifica della salute delle cellule e la densità. </li><li> Al termine, le celle a filo nel contenitore originale. Pulire la cassetta eseguendo 50 ml di etanolo al 70% seguita da 50 ml di acqua sterile calda attraverso il tubo. </li><li> Assicurarsi cassette e tubi sono aptamente puliti tra usi. </li></ol></li><li> Incubare le cellule per 24 ore a 37 ° C in un umidificata al 5% di CO 2 incubatore. </li></ol> 3 Preparazione di Viral curva standard <ol><li> Preparare una curva standard di VSVΔ51-Fluc in DMEM senza siero tale che la concentrazione finale delle unità formanti placca (PFU) per ml dopo il trasferimento su cellule Vero è la seguente: 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, e 10 1 pfu / ml. Preparare 50 ml di ciascuna concentrazione per piastra di cellule Vero, più un ulteriore 10%. NOTA: Titer del virus magazzino luciferasi dovrà essere valutata in modo classico 10, al fine di generare una curva standard che permetterà per la quantificazione assoluta. Altrimenti relativa quantificazione può essere raggiunto senza informazioni precise titolo fissando arbitrariamente titolo virale basatasui gradini di diluizione. Ad esempio la prima diluizione della curva standard può essere impostato su 10 8 unità virali e la diluizione 1/10 testo seguente 10 7 e così via. In questo contesto, si dovrebbe esprimere i valori come un fold-variazione rispetto ad un campione predeterminato come quantificazione assoluta non sarà preciso. </li></ol> 4 Trasferimento di surnatante del campione su celle di consenso <ol><li> Controllare cellule Vero placcato nella piastra a 96 pozzetti con fondo trasparente sotto un microscopio ottico per confermare che monostrati sono almeno il 95% confluenti. </li><li> Trasferire 25 ml di campione di surnatante su cellule Vero seminate in piastre bianche pareti. Non trasferire surnatanti nelle 2 colonne designate per le curve standard. NOTA: Questo può essere fatto contemporaneamente per tutti i pozzetti su un unico piatto utilizzando un gestore di liquido a 96 canali. </li><li> Utilizzando una pipetta multicanale a 8 o 12 canali, aggiungere 25 ml di ogni diluizione della curva standard preparata al punto 3 per le cellule Vero inle 2 colonne designate. </li><li> Piastre di centrifugazione per 5 min a 430 xg a temperatura ambiente. </li><li> Incubare per 5 ore a 37 ° C in un umidificata al 5% di CO 2 incubatore. </li></ol> 5 Valutazione della vitalità cellulare <ol><li> NOTA: Quando si avvia da surnatanti ottenuti da esperimenti di infezione fatto in piastre a 96 pozzetti chiari, la redditività del campione può essere valutata prima di quantificazione con un indicatore di vitalità cellulare colorante come resazurina. </li><li> Aggiungere resazurina in quantità pari al 10% del volume in ciascun pozzetto della piastra a 96 pozzetti di campioni contenenti virus e cellule. Includi cellulare solo controlla e di controllo solo media per determinare rispettivamente i valori per il 100% e 0% vitalità. </li><li> Dopo 2-4 ore (tempo di incubazione varia a seconda del tipo di cellula e sulla concentrazione di disponibili in commercio o in polvere ricostituito del colorante), leggere e registrare il segnale utilizzando un lettore di piastre a fluorescenza (eccitazione 530-560, 590 emissione). Relazione viabili cellularety per un campione in base alla formula relativa attività metabolica = ((segnale campione di prova - segnale di controllo negativo) / (cellulare segnale di controllo solo - segnale di controllo negativo)) x 100% </li></ol> 6 Preparazione del substrato luciferina <ol><li> 30 minuti prima della fine dell&#39;incubazione 5 ore, preparare la luciferina per ottenere una soluzione / ml 2 mg sterile tampone fosfato salino (PBS). Preparare 2,5 ml per piastra più un extra di 2 ml. Proteggere la soluzione dalla luce. NOTA: La luciferina può anche essere preparato in precedenza in giornata e conservato a 4 ° C fino al momento dell&#39;uso. </li></ol> 7 Lettura bioluminescenza <ol><li> Dopo il periodo di incubazione di 5 ore, aggiungere 25 ml di luciferina in ciascun pozzetto di cellule Vero nei piatti solidi bianchi. Aggiungere luciferina manualmente o con un dosatore automatico integrato nel luminometro. NOTA: L&#39;uso di uno strumento con unico punto singolo e end legge può richiedere l&#39;aggiunta di una luciferasi lisi BUF compatibilefer prima di aggiungere il substrato luciferina di migliorare la coerenza. </li><li> Leggere le piastre con i seguenti parametri: <ol><li> Agitare per 5 sec. </li><li> Attendere 30 sec. </li><li> Leggi luminescenza ad un appropriato valore di sensibilità / esposizione fisso. Se l&#39;opzione è disponibile sullo strumento, utilizzare multi-point legge (ad esempio, matrice 3 x 3) per migliorare la precisione. </li></ol></li><li> Registrare e salvare l&#39;intensità bioluminescente quantificato. </li><li> Se un distributore automatico è stato utilizzato per aggiungere luciferina spurgare la luciferina e adescare le linee con acqua sterile tiepida. </li></ol> Analisi dei dati 8. <ol><li> Per ottenere risultati accurati, risolvere la regressione non lineare per generare un&#39;equazione Hill dalle curve standard. Applicare questa equazione per i campioni titolato al fine di calcolare le unità di espressione virale. Alcuni virus o situazioni possono produrre una curva standard con una relazione lineare; in questo caso per risolvere una equazione lineare. </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.

L&#39;approccio basato luciferasi-qui descritta fornisce una serie di vantaggi rispetto ad altri metodi esistenti, compresa la sua facilità, rapidità, minima necessità di attrezzature, e relativamente a basso costo. Un contributo fondamentale a questo è quello di evitare una fase di diluizione seriale. Tuttavia, i derivati ​​di serie a base di diluizione, di questo protocollo sono certamente fattibile e sono stati recentemente utilizzati per valutare-luciferasi che esprimono Ebola titoli in uno schermo antivirale high-throughput 11. Mentre intrinsecamente più tempo e più costoso, tali adattamenti possono fornire una maggiore gamma dinamica per la quantificazione virale quando necessario. Oltre ad essere particolarmente adatto per la valutazione titoli virali nel contesto degli schermi high-throughput, il nostro metodo di quantificazione virale base di uno stadio luciferasi genera stime accurate di virioni infettivi nel caso di replicare virus. Inoltre, letture di luminescenza insieme ai dati di citotossicità dello stesso esperimento danno una piùquadro completo l&#39;effetto delle condizioni sperimentali su cellule bersaglio, che è particolarmente utile nel contesto di virus oncolitici e schermi di droga. L&#39;esempio qui illustrato utilizza un replicante virus RNA a singolo filamento negativo; Tuttavia, questo protocollo può essere adattato ad un numero di replicare e non replicanti virus con pochi adattamenti protocollo minori. Questo include virus a DNA come vaccinico, HSV, e AAV (vedi Figura 5). I campioni infettati da virus intracellulari, come il virus vaccinico, ad esempio, richiedono un passo virus-release prima di quantificazione (ad esempio, almeno un ciclo gelo-disgelo). Quando si applica questa tecnica ad altri virus, è necessario ottimizzare il tempo di incubazione dal trasferimento del surnatante virale o lisato alla lettura delle piastre dal luminometro. Questo parametro dipende principalmente dal ciclo di replicazione del virus nella linea cellulare permissiva e la forza del promoter guida espressione luciferasi. Questo è fatto meglio usando la curva standard completa nella fase di ottimizzazione. Per fare questo, si deve infettare la linea cellulare permissiva appropriata con varie repliche della curva standard preparata e leggere ogni replica in un momento diverso punti post-trasferimento. Idealmente, un punto di tempo di incubazione è scelto che conduce ad una relazione lineare tra LOG (RLU) e LOG (titolo) che attraversa il campionario titolo atteso. Per VSVΔ51-Fluc, questo è in genere da 10 a 4 pfu / ml -10 7 pfu / ml per un tempo di incubazione 5 ore. Se titoli inferiori o superiori sono attesi da campioni, si può semplicemente aumentare o ridurre il tempo di incubazione, rispettivamente. In alternativa, i campioni possono essere diluiti per rientrare nel campo di quanto avviene tipicamente per ELISA. Come accennato in precedenza, questo metodo è adatto per eseguire droga schermi high-throughput utilizzando librerie di droga, come la maggior parte delle operazioni possono essere automatizzate. Le cellule possono essere placcati efficiently utilizzando un dispensatore di micropiastre automatico, la biblioteca farmaco può essere aggiunto utilizzando un gestore di liquido a 96 canali, virus può essere aggiunto utilizzando un dispenser di micropiastre e piastre leggere utilizzando un luminometro automatico. In teoria, questo può anche essere adattato a 384 pozzetti o formati più piccoli; tuttavia, la limitazione a tal fine è il numero di cellule che possono essere placcato, dato minor numero di cellule porta ad una gamma più ristretta nella linearità del LOG (RLU) a LOG (Titer) rapporto. Infine, la valutazione della vitalità cellulare mediante resazurina o altri coloranti metabolici può essere facilmente integrato nel flusso di lavoro, consentendo la discriminazione di composti citotossici in schermi antivirali o identificazione di composti che portano alla uccisione sinergica in combinazione con virus 12. Tuttavia, le limitazioni di questo metodo includono il requisito di un virus transgene che esprime la luciferasi, che non è sempre possibile, e la disponibilità di una linea cellulare permissiva sufficientemente. Tuttavia, è probabile possibile adattareil metodo usato con altri geni reporter (per esempio, GFP), se il metodo di quantificazione giornalista ha una linearità adatto e rapporto segnale rumore. In generale, il metodo high-throughput descritto può essere modificato per soddisfare molti virus diversi e su misura per diverse applicazioni.

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.

Una sintesi del flusso che descrive il metodo ad alta produttività è illustrato in Figura 1. Figura 2 mostra i risultati di una tipica curva standard ofVSVΔ51-Fluc. Quattro parametro analisi di regressione non lineare generato una trama collina da cui ingresso sconosciuto pfu (stima del titolo virale) possono essere interpolati. Questi titoli stimati sono denominate unità di espressione virali (VEU). Figura 3A mostra VEUS e titoli ottenuti eseguendo un saggio di targa di serie con gli stessi campioni 10. I campioni provenivano da un esperimento in cui sono stati utilizzati vari prodotti chimici per migliorare la replicazione e la diffusione di VSVΔ51-Fluc in 786-0 cellule. VEUS interpolato dalla curva standard deve essere moltiplicato per un fattore che è basato sulla diluizione del campione surnatanti stati trasferiti a cellule Vero (in questo caso, il fattore di diluizione è 5). La correlazione lineare tra titoli e VEU è mostrato in Figura 3B </strong&gt; Con un R 2 di 0,8083 e r punteggio di un Pearson di 0,899 (p &lt;0,0001). Nella Figura 4, i dati tipici citotossicità cellulo ottenuti da un test metabolico è indicata per 786-0 cellule trattate con sostanze chimiche prima di infezione da VSVΔ51-Fluc. Infine, la Figura 5 mostra le curve tipiche standard ottenute con vari virus (Herpes Simplex Virus (HSV), il virus vaccinico, e virus adeno-associato (AAV), tutti esprimono luciferasi Firefly) e descrive i tempi di incubazione e cicli di gelo e disgelo, come richiesto. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> Figura 1 Flusso di lavoro di high-throughput titolazione virus e test di citotossicità utilizzando VSV Δ 51-Fluc. (A) Seed 2,5 x 10 4 cellule Vero per pozzetto (100 ml) e incubare a 37 ° C. (B) 24hr trasferimento successivamente 25 ml di curva standard su cellule Vero (2 colonne per piastra). (C) Trasferire 25 ml di campioni per essere titolati a rimanenti cellule Vero. Piastre di centrifuga e incubare a 37 ° C. (D) per un importo pari al 10% del volume del bene, aggiungere il reagente resazurina alla piastra contenente i campioni originali. Incubare le piastre a 37 ° C. (E) Dopo 3 ore, leggere e registrare la fluorescenza e valutare la citotossicità. (F) Dopo 5 ore, aggiungere 25 ml di soluzione di 2 mg / ml di luciferina in ogni pozzetto di cellule Vero. Leggi luminescenza e calcolare Unità Espressione virali. <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> Figura 2 Previsto curva standard di VSV Δ 51-Fluc. Espressione luciferasi me eraasured 5 hr trasferimento, dopo il surnatante in cinque punti diversi all&#39;interno di un pozzo con un luminometro e bioluminescenza è stato espresso in media unità di luce relativa (RLU). RLU media è stata rilevata in entrata noto pfu / ml per risolvere la regressione non lineare e generare una equazione di Hill. La media delle due curve ripetute e barre di errore standard sono riportati (R 2 = 0,9993). <img alt="Figura 3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> Figura 3 Confronto di titoli test placca standard con quelli ottenuti con il metodo alto throughput. (A) titoli virali in pfu / ml ottenuti dal saggio di placca standard su cellule Vero sono stati confrontati con titoli virali calcolati (VEU / ml) dagli stessi campioni titolati utilizzando il saggio high-throughput luciferasi. (B) relazione lineare tra VEU / ml e titolo ottenuto tramite p di serietest Laque. Sono mostrati curva di regressione lineare e coefficiente di determinazione (R 2). <img alt="Figura 4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> Figura 4 Esempio di vitalità. Redditività del campione prima del trasferimento surnatante su cellule Vero è stata determinata valutando l&#39;attività metabolica cellulare usando una soluzione resazurina disponibile in commercio. Valori di fluorescenza Raw sono stati normalizzati a quella di non trattati, pozzi non infetti. <img alt="Figura 5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> Figura 5 Previsto curve standard con HSV, Vaccinia, e virus AAV. Espressione luciferasi è stata misurata in cinque punti diversi all&#39;interno di un pozzo con un luminometro e bioluminescenza è stato espresso in rela medigruppi ottici tivi (RLU). (A) curva standard HSV è stato aggiunto su cellule Vero placcato 24 ore prima ad una densità di 2,5 x 10 4 cellule per pozzetto (100 ml) e la misurazione della luciferasi è stata fatta 17 ore dopo il trasferimento surnatante (R 2 = 0,9489, n = 1). Un&#39;equazione Hill è stata generata risolvendo la regressione non lineare. Curva standard (B) vaccinico stato aggiunto su cellule Vero placcato 24 h prima una densità di 2,5 x 10 4 cellule per pozzetto (100 microlitri) e incubate per 2,5 ore a 37 ° C, dopo di che sono state prese le misure luciferasi (R 2 = 0,9892). Un&#39;equazione lineare è stata generata risolvendo per la regressione lineare. (C) AAV curva standard è stato aggiunto su cellule di carcinoma polmonare umano (A549) placcato 24 ore prima ad una densità di 2,5 x 10 4 cellule per pozzetto (100 microlitri) e la misurazione luciferasi è stata fatta 24 ore dopo l&#39;infezione. Un&#39;equazione Hill è stata generata risolvendo la regressione non lineare. La mediadi sono mostrati cinque curve ripetute e barre di errore standard (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.

High-throughput Titolazione della luciferasi che esprimono ricombinanti virus

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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Biology

12.8K Views.  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.

High-throughput iperespressione schermo lievito plasmidi

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

Ricerca

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.

High-throughput per l&#39;analisi Saccarificazione materiali lignocellulosici

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.

High-throughput Purificazione di affinità-tagged proteine ​​ricombinanti

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 microiniezioni di Sea Urchin zigoti

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 espressione quantitativa e purificazione Applicata al ricombinante Disulfide ricchi di Venom proteine ​​prodotte in<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.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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.

Identificazione delle coppie Kinase-substrato Usando 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 spese 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.

-High Throughput, Multi-Image Cryohistology di mineralizzati tessuti

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