Name: high-throughput titration of luciferase-expressing recombinant viruses
Uploaded: 2014-09-19T15:00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about High-throughput Titration of Luciferase-expressing Recombinant Viruses at JoVE.com

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

1. Sample Preparation
<ol>
	<li>Obtain samples containing luciferase-expressing virus (herein VSV&#8710;51-Fluc as an example) for titration and transfer into 96-well plates. Alternately, perform infections in 96-well tissue culture plates and use supernatants directly.</li>
	<li>Leave two columns untreated for the inclusion of standard curves. For experiments done directly in 96-well plates, titer at the end of the experiment (40 hr&#160;post-infection) or store at -80 &#176;C and titer at a later date.</li>
</ol>
2. Preparation of Permissive Cells for Virus Titration
<ol>
	<li>24 hr&#160;prior to titering, prepare a suspension of Vero cells at a concentration of 2.5 x 105 cells/ml&#160;in Dulbecco&#8217;s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 30 mM HEPES, and 1% penicillin/streptomycin. NOTE: Although this protocol uses Vero cells, any suitable permissive cell line for VSV infection could be used.</li>
	<li>Seed 2.5 x 104 cells (100 &#956;l) in 96-well white solid flat-bottom plates using a microplate dispenser.
	<ol>
		<li>Prepare 12 ml&#160;of cell suspension per plate plus 5 ml&#160;for priming.</li>
		<li>Clean microplate dispenser cassette by flushing the tubing with 50 ml&#160;of sterile water.</li>
		<li>Fill the microplate dispenser lines with cell suspension and let 5 ml&#160;of cell suspension flow through.</li>
		<li>Select a program that will dispense 100&#160;&#956;l in each well of a white-walled 96-well plate. Dispense, and repeat as necessary for additional plates. Also seed a few wells in a 96-well clear flat-bottom plate if opaque-bottom white-walled plates are used for verification of cell health and density.</li>
		<li>When finished, flush cells back into original container. Clean the cassette by running 50 ml&#160;of 70% ethanol followed by 50 ml&#160;of warm sterile water through the tubing.</li>
		<li>Make sure cassette and tubing are appropriately cleaned between uses.</li>
	</ol>
	</li>
	<li>Incubate cells for 24 hr at 37 &#176;C in a humidified 5% CO2 incubator.</li>
</ol>
3. Preparation of Viral Standard Curve
<ol>
	<li>Prepare a standard curve of VSV&#8710;51-Fluc in serum-free DMEM such that the final concentration of plaque forming units (pfu) per ml&#160;after transfer onto Vero cells is as follows: 108 pfu/ml, 107 pfu/ml, 106 pfu/ml, 105 pfu/ml, 104 pfu/ml, 103 pfu/ml, 102 pfu/ml, and 101 pfu/ml. Prepare 50 &#956;l of each concentration per plate of Vero cells plus an extra 10%. 
	NOTE: Titer of the luciferase virus stock will need to be assessed in a classical way10 in order to generate a standard curve that will allow for absolute quantification. Otherwise relative quantification can be achieved without precise titer information by arbitrarily setting viral titer based on dilution steps. For example the first dilution of the standard curve may be set to 108 viral units and the following 1/10 dilution to 107 and so on. In this context, one should express values as a fold-change compared to a pre-determined sample as absolute quantification will not be accurate.</li>
</ol>
4. Transfer of Sample Supernatants onto Permissive Cells
<ol>
	<li>Check Vero cells plated in the clear-bottom 96-well plate under a light microscope to confirm that monolayers are at least 95% confluent.</li>
	<li>Transfer 25 &#956;l of sample supernatant onto Vero cells seeded in white-walled plates. Do not transfer supernatants into the 2 columns designated for standard curves. NOTE: This can be done simultaneously for all wells on a single plate using a 96-channel liquid handler.</li>
	<li>Using an 8- or 12-channel multi-channel pipettor, add 25 &#956;l of each dilution of the standard curve prepared in Step 3 to the Vero cells in the 2 designated columns.</li>
	<li>Centrifuge plates for 5 min at 430 x g at RT.</li>
	<li>Incubate for 5 hr at 37 &#176;C in a humidified 5% CO2 incubator.</li>
</ol>
5. Assessment of Cell Viability
<ol>
	<li>NOTE: When starting from supernatants obtained from infection experiments done in clear 96-well plates, sample viability can be assessed prior to quantification with a cell viability indicator dye such as resazurin.</li>
	<li>Add resazurin in an amount equal to 10% of the volume in each well of the 96-well plate of samples containing virus and&#160;cells. Include cell-only controls as well as media-only control to determine values for 100% and 0% viability respectively.</li>
	<li>After 2-4 hr (incubation time will vary depending on cell type and on the concentration of commercially available or reconstituted powder of the dye), read and record the signal using a fluorescence plate reader (530-560 excitation, 590 emission). Report cell viability for a sample according to the formula Relative Metabolic Activity = ((Test sample signal - negative control signal) / (Cell only control signal - negative control signal)) x 100%</li>
</ol>
6. Preparation of the Luciferin Substrate
<ol>
	<li>30 min&#160;before the end of the 5 hr incubation, prepare the luciferin to obtain a 2 mg/ml&#160;solution in sterile phosphate buffered saline (PBS). Prepare 2.5 ml&#160;per plate plus an extra 2 ml. Protect solution from light. NOTE: The luciferin may also be prepared earlier in the day and stored at 4 &#176;C until use.</li>
</ol>
7. Reading Bioluminescence
<ol>
	<li>After the 5 hr incubation period, add 25 &#956;l of luciferin to each well of Vero cells in the white solid plates. Add luciferin manually or with an automated dispenser integrated in the luminometer. NOTE: The use of an instrument with only single and end point reads may require the addition of a luciferase compatible lysis buffer prior to adding the luciferin substrate to improve consistency.</li>
	<li>Read the plates with the following parameters:
	<ol>
		<li>Shake for 5 sec.</li>
		<li>Wait 30 sec.</li>
		<li>Read luminescence at an appropriate fixed sensitivity / exposure value. If the option is available on the instrument, use multi-point reads (e.g., 3 x 3 matrix) to improve accuracy.</li>
	</ol>
	</li>
	<li>Record and save the quantified bioluminescent intensity.</li>
	<li>If an automated dispenser was used to add luciferin purge the luciferin and prime the lines with warm sterile water.</li>
</ol>
8. Data Analysis
<ol>
	<li>For accurate results, solve the non-linear regression to generate a Hill equation from the standard curves. Apply this equation to the titered samples to calculate viral expression units. Some viruses or situations may produce a standard curve with a linear relationship; in this case solve for a linear equation.</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.

The luciferase-based approach described here provides a number of advantages over other existing methods including its ease, quickness, minimal equipment need, and relatively low cost. A key contributor to this is the avoidance of a serial dilution step. Nonetheless, serial dilution-based derivatives of this protocol are certainly feasible and were recently used to assess luciferase-expressing Ebola titers in a high-throughput antiviral screen11. While inherently more time consuming and more expensive, such adaptations may provide greater dynamic range for viral quantification when necessary. In addition to being particularly well suited for evaluating viral titers in the context of high-throughput screens, our one-step luciferase based viral quantification method generates accurate estimates of infectious virions in the case of replicating viruses. Furthermore, readouts of luminescence along with cytotoxicity data from the same experiment give a more complete picture of the effect of the experimental conditions on target cells, which is particularly useful in the context of oncolytic viruses and drug screens.
The example illustrated here uses a replicating negative single strand RNA virus; however, this protocol can be adapted to a number of replicating and non-replicating viruses with a few minor protocol adjustments. This includes DNA viruses such as Vaccinia, HSV, and AAV (see Figure 5). Samples infected with intracellular viruses, such as Vaccinia virus for example, require a virus-release step prior to quantification (e.g., at least one freeze-thaw cycle). When applying this technique to other viruses, it is necessary to optimize the incubation time from the transfer of the viral supernatant or lysate to the reading of the plates by the luminometer. This parameter will depend mainly on the replication cycle of the virus in the permissive cell line and the strength of the promoter driving luciferase expression. This is best done by using the full standard curve in the optimization step. To do this, one must infect the appropriate permissive cell line with various replicates of the prepared standard curve and read each replicate at a different time points post-transfer. Ideally, an incubation time point is chosen that leads to a linear relationship between LOG(RLU) and LOG(titer) spanning the expected sample titer range. For VSV&#8710;51-Fluc, this is typically from 104 pfu/ml&#160;-107 pfu/ml&#160;for a 5 hr incubation time. If lower or higher titers are expected from samples, one can simply increase or reduce the incubation time respectively. Alternately, samples may be diluted to fall within the range much as is done typically for ELISA.
As mentioned above, this method is well suited to perform high-throughput drug screens using drug libraries as most of the steps can be automated. Cells can be plated efficiently using an automated microplate dispenser, the drug library can be added using a 96-channel liquid handler, virus can be added using a microplate dispenser and plates read using an automated luminometer. In theory, this can also be adapted to 384-well or smaller formats; however, the limitation to this end is number of cells that can be plated, given fewer cells leads to a narrower range in the linearity of the LOG(RLU) to LOG(Titer) relationship. Finally, assessment of cell viability using resazurin or other metabolic dyes can be easily incorporated in the workflow, allowing for discrimination of cytotoxic compounds in antiviral screens or identification of compounds that lead to synergistic killing in combination with viruses12. Nevertheless, limitations of this method include the requirement of a luciferase transgene expressing virus, which is not always possible, and the availability of a sufficiently permissive cell line. However, it is likely possible to adapt the method for use with other reporter genes (e.g., GFP) provided the reporter quantification method has a suitable linearity and signal to noise ratio. Overall, the described high-throughput method can be modified to suit many different viruses and tailored to diverse applications.

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.

A summary of the workflow describing the high-throughput method is illustrated in Figure 1. Figure 2 shows the results of a typical standard curve ofVSV&#8710;51-Fluc. Four-parameter non-linear regression analysis generated a Hill plot from which unknown input pfu (estimate of viral titer) can be interpolated. These estimated titers are termed viral expression units (VEU). Figure 3A shows VEUs and titers obtained by performing a standard plaque assay with the same samples10. Samples originated from an experiment where various chemicals were used to enhance the replication and spread of VSV&#8710;51-Fluc in 786-0 cells. VEUs interpolated from the standard curve must be multiplied by a factor which is based on the dilution of sample supernatants being transferred on to Vero cells (in this case, the dilution factor is 5). The linear correlation between titers and VEU is shown in Figure 3B with an R2 of 0.8083 and a Pearson&#8217;s r score of 0.899 (p &#60;0.0001). In Figure 4, typical cell cytotoxicity data obtained from a metabolic assay is shown for 786-0 cells treated with chemicals prior to infection with VSV&#8710;51-Fluc. Finally, Figure 5 shows typical standard curves obtained with various viruses (Herpes Simplex Virus (HSV), Vaccinia virus, and Adeno-Associated Virus (AAV), all expressing Firefly luciferase) and describes incubation times and freeze-thaw cycles, as required.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig1highres.jpg" width="500" /> 
Figure 1. Workflow of high-throughput virus titering and cytotoxicity assay using VSV&#8710;51-Fluc. (A) Seed 2.5 x 104 Vero cells per well (100 &#956;l) and incubate at 37 &#176;C. (B) 24 hr later transfer 25 &#956;l of standard curve on to Vero cells (2 columns per plate). (C) Transfer 25 &#956;l&#160;of samples to be titered on to remaining Vero cells. Centrifuge plates and incubate at 37 &#176;C. (D) In an amount equal to 10% of the volume in the well, add resazurin reagent to the plate containing the original samples. Incubate plates at 37 &#176;C. (E) After 3 hr, read and record fluorescence and assess cytotoxicity. (F) After 5 hr, add 25 &#956;l&#160;of 2 mg/ml&#160;solution of luciferin to each well of Vero cells. Read luminescence and calculate Viral Expression Units.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig2highres.jpg" width="500" /> 
Figure 2. Expected standard curve of VSV&#8710;51-Fluc. Luciferase expression was measured 5&#160;hr post supernatant transfer at five different points within a well using a luminometer and bioluminescence was expressed in mean relative light units (RLU). Mean RLU was plotted against known input pfu/ml&#160;to solve the non-linear regression and generate a Hill equation. The average of two replicate curves and standard error bars are shown (r2= 0.9993).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig3highres.jpg" width="500" /> 
Figure 3. Comparison of standard plaque assay titers with those obtained by high-throughput method. (A) Viral titers in pfu/ml&#160;obtained by standard plaque assay on Vero cells were compared with calculated viral titers (VEU/ml) from the same samples titered using the high-throughput luciferase assay. (B) Linear relationship between VEU/ml&#160;and titer obtained via standard plaque assay. Linear regression curve and coefficient of determination (R2) are shown.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig4highres.jpg" width="500" /> 
Figure 4. Sample viability. Sample viability prior to supernatant transfer onto Vero cells was determined by assessing cellular metabolic activity using a commercially available resazurin solution. Raw fluorescence values were normalized to that of untreated, uninfected wells.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51890/51890fig5highres.jpg" width="500" /> 
Figure 5. Expected standard curves with HSV, Vaccinia, and AAV viruses. Luciferase expression was measured at five different points within a well using a luminometer and bioluminescence was expressed in mean relative light units (RLU). (A) HSV standard curve was added onto Vero cells plated 24 hr earlier at a density of 2.5 x 104 cells per well (100 &#956;l) and luciferase measurement was made 17 hr post supernantant transfer (R2= 0.9489, n=1). A Hill equation was generated by solving the non-linear regression. (B) Vaccinia standard curve was added onto Vero cells plated 24 hr earlier a density of 2.5 x 104 cells per well (100 &#956;l) and incubated for 2.5 hr at 37 &#176;C, after which luciferase measurements were taken (R2= 0.9892). A linear equation was generated by solving for the linear regression. (C) AAV standard curve was added onto human lung carcinoma cells (A549) plated 24 hr&#160;earlier at a density of 2.5 x 104 cells per well (100 &#956;l) and luciferase measurement was made 24 hr post infection. A Hill equation was generated by solving the non-linear regression. The average of five replicate curves and standard error bars are shown (R2= 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.

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

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.

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

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.

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.

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.

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.

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.

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.

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.


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.

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.

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