The authors thank Dr. M. Matrosovich for providing the parent MDCK and MDCK-SIAT1 cell lines and Roche Pharmaceuticals for supplying the oseltamivir carboxylate.We also appreciate Dr. Anne Weston for valuable suggestions on the manuscript. This study was dependent upon the valued collaboration of the WHO National Influenza Centres and the WHO CCs within the WHO GISRS, who provided the influenza viruses used. This work was funded by the Medical Research Council through Programme U117512723.

NOTE: The biosafety level (BSL) for the following protocol is BSL 2 for seasonal influenza viruses and BSL 3+ for potential pandemic influenza viruses. Viral titration is required in advance of a neutralization experiment to decide the viral concentration of the viral control (VC).
1. Virus Titration
NOTE: Depending on the number of viruses and the number of duplicates, a well plate can be set up with the following flexibilities: (1) The viral dilution can be arranged along either the rows or the columns (Figure 1). Each virus should occupy a separate row/column. (2) The viral dilution increment is flexible, but it should start with the highest viral concentration at the top-left corner. (3) There is no restriction on the number of duplicates.
NOTE: Madin Darby canine kidney (MDCK) and MDCK-SIAT1 (SIAT) cells were kindly provided by Dr. M. Matrosovich, Marburg, Germany10. SIAT cells are MDCK cells stably transfected with the human CMP-N-acetylneuraminate: &#946;-galactoside &#945;-2,6-sialyltransferase gene for enhanced expression of sialic acid (SA) &#945;2-6Gal-terminated oligosaccharides.
<ol>
	<li>Aliquot sufficient MDCK cells or MDCK-SIAT cells8 into 96-well plates (200 &#181;l/well). Incubate the cells at 37 &#176;C with 5% CO2 for 2 or 3 days to reach confluence. Propagate the cells in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (100 U/ml penicillin and 100 &#181;g/ml streptomycin). Incubate the SIAT cells at 37 &#176;C with 5% CO2 and 1 mg/ml G418 sulphate. 
	NOTE: The dilution factor is dependent upon experience and the cell line used. The cell monolayer must be confluent (i.e., no cell wall or visible outline for MDCK cells and a tightly-packed layout for MDCK-SIAT1 cells) at the time of virus inoculation. Examples of dilutions based on the subculture of confluent flasks of MDCK or MDCK-SIAT1 cells are given in Table 1.</li>
	<li>Wash the cells 3 times with 200 &#181;l of virus growth medium (VGM) per well. Sterilize the multiwall plate washer with 70% EtOH for 30 min, and then rinse it in sterile phosphate-buffered saline (PBS) or VGM before the wash.</li>
	<li>Aspirate the VGM and immediately add 50 &#181;l of VGM to each well.</li>
	<li>Add 900 &#181;l of VGM to each of 6 sterile tubes (or fewer, depending upon the number of test viruses and duplicates). Pipette 100 &#181;l of virus into the first tube, mix well, and serially dilute, changing the tips between each dilution. Repeat for each virus to be titrated. 
	NOTE: This will give virus dilutions of 10-1 to 10-6.</li>
	<li>Add 50 &#181;l of each virus dilution, starting at the highest dilution (1 x 10-5 in Figure 1), to duplicated wells (Columns 9 and 10 in Figure 1) of a 96-well plate.</li>
	<li>Place the plate at 37 &#176;C for 2-3 hr to allow the virus to infect the cells. 
	NOTE: A 2 to 3 hr incubation is necessary to ensure that the viruses in the inoculum have enough time to reach the monolayer.</li>
	<li>Prepare the overlay (10 ml/plate) 
	NOTE: The overlay consists of 5 ml of 2x DMEM, Trypsin (2 &#181;g/ml final concentration), 20 &#181;l of 1 mg/ml stock, and 5 ml of cellulose cotton linters (see the Materials List).</li>
	<li>Remove the inoculum.</li>
	<li>Add the overlay (200 &#181;l to each well). Incubate overnight at 37 &#176;C, UNDISTURBED. 
	NOTE: The virus budding takes place after approximately 4 hr, so it is important that the plate is not disturbed after this time.</li>
	<li>Aspirate the overlay from the wells.</li>
	<li>Add ice-cold 4% paraformaldehyde in PBS A (200 &#181;l/well). Place them at 4 &#176;C for 30 min or at room temperature for 20 min. 
	NOTE: PBS A is natural-pH phosphate-buffered saline with 10 g of NaCl, 0.25 g of KCl, 1.437 g of Na2HPO4, 0.25 g of KH2PO4, and 1 L of distilled water (see the Materials List). 
	NOTE: Paraformaldehyde is harmful when inhaled and can cause burns if swallowed. There is also limited evidence of a carcinogenic effect, and it may cause sensitization after skin contact.</li>
	<li>Aspirate the paraformaldehyde and wash the plates twice with PBS A (200 &#181;l/well).</li>
	<li>Store the plates in PBS A at 4 &#176;C for future use, or carry on with the following steps.</li>
	<li>Add permeabilization buffer (100 &#181;l/well). Leave it at room temperature for 30 min.</li>
	<li>Wash plate twice with PBS A (200 &#181;l/well).</li>
	<li>Add 50 &#181;l of the first antibody, mouse MAb against influenza type A (1:1,000 in ELISA Buffer), per well. Incubate it at room temperature for 1 hr (or 4 &#176;C overnight), with shaking.</li>
	<li>Wash it 3 times in 100 &#181;l of wash buffer (0.05% Tween 80 in PBS A, v/v) per well. Incubate it at room temperature for 5 min between washes. Alternately, wash it 3 times without incubation using 300 &#181;l per well.</li>
	<li>Add 50 &#181;l of the second antibody, goat anti-mouse IgG (H+L) HRP conjugate (1:1,000 in ELISA Buffer), per well. Incubate it at room temperature for 1 hr, with shaking.</li>
	<li>Wash it 3 times as described in step 1.17.</li>
	<li>Add the substrate (50 &#181;l/well). Incubate it at room temperature for 30 min or until the development of a blue color is clearly visible.</li>
	<li>To stop the reaction, wash the plate twice with 200 &#181;l of distilled water per well, incubating at room temperature for 2 -3 min between the washes.</li>
	<li>Air-dry the plate and store it in a dark place (e.g., wrap it in aluminum foil).</li>
</ol>
2. Titration Quantitation
<ol>
	<li>Place a well plate in the scanning area of a flatbed scanner, as shown in Figure 2a. Use the L-shape position limit to ensure an optimum and repeatable imaging location. 
	NOTE: It is possible to image two well plates in one scan.</li>
	<li>Scan an image. 
	NOTE: The settings are shown in Table 2.</li>
	<li>Run the &#34;Wellplate Reader&#34; software to calculate the required virus concentration (Figure 3).
	<ol>
		<li>Click the &#34;Load image&#34; button to load an image. Slide the red bar in the histogram of the &#34;Global Threshold&#34; tab to adjust the sampling threshold. Click the &#34;Update&#34; button to examine the effect on the image.</li>
		<li>Tick the &#34;Calculate Neutralization/Titration&#34; box. Click the &#34;Sampling&#34; button to quantify the ICP. Click the &#34;Save&#34; button to save the sampling results when prompted. 
		NOTE: After the sampling process, a new window called &#34;Neutralization &#38; Titration Calculation&#34; appears automatically if the &#34;Calculate Neutralization/Titration&#34; box is ticked.</li>
		<li>Load or input the well-plate definition map. Indicate the threshold (e.g., 30%) in &#34;Titration: Optimal Virus Population (%)&#34;. Select the &#34;Titration Process&#34; tab to calculate the titration results. Check the titration results. Click the &#34;Save &#38; Close&#34; button to save the titration results. 
		NOTE: Refer to Supplementary S1 for more detailed instruction on the software. A copy of the bespoke software is available upon request. 
		NOTE: Typically, the best virus dilution for the plaque-reduction assay yields around 50% (20-85%) ICP of total cells within each well11.</li>
	</ol>
	</li>
</ol>
3. Virus Neutralization
NOTE: Calculate the number of plates required for the neutralization assay. Each plate can accommodate one virus and a number of antisera.
NOTE: Depending on the number of antisera and the number of duplicates, a well plate can be set up with the following flexibilities: (1) The serum dilution can be arranged along either row or a column (Figure 4). Each serum should occupy a separate row or column. (2) The increment of serum dilution is flexible, but it should start with the highest serum concentration at the top-left corner of a plate. (3) The number of duplicates balances the number of antisera. More duplicates can help to smooth out experimental variations. (4) The number of the VC and the number of the cell control (CC) duplicates are flexible, but they should also be in separate rows or columns. It is expected that the number of VC duplicates is significantly greater than that of the antisera.
<ol>
	<li>Prepare the cell monolayer, as in steps 1.1-1.3, 2 or 3 days in advance.</li>
	<li>Add 50 &#181;l of VGM to each well of the plate.</li>
	<li>Use Columns 11 and 12 for VC and CC, respectively. Add 50 &#181;l aliquots of 1:20 receptor-destroying enzyme (RDE)-treated serum to the first row (A) of Columns 1-10. 
	NOTE: For each virus and serum combination, the assay is performed in duplicate, so one plate may, for example, contain one virus tested against five antisera.</li>
	<li>Perform 2-fold serial dilutions by transferring 50 &#181;l from Row A to Row H (Columns 1-10 in Figure 4) and discarding 50 &#181;l from Row H.</li>
	<li>Add 50 &#181;l of diluent to each well of the CC column (Column 12 in Figure 4).</li>
	<li>Add 50 &#181;l of the virus to each well of the plate, except for the CC column (Columns 1-11, Rows A-H in Figure 4).</li>
	<li>Incubate it at 37 &#176;C for 2-3 hr. Remove the inoculum. Add the overlay (200 &#181;l) to each well. Incubate it overnight at 37 &#176;C, UNDISTURBED. Fix and stain the plates as for the virus titration (steps 1.11-1.22).</li>
</ol>
4. Neutralization Quantification
<ol>
	<li>Place a well plate in the scanning area of a flatbed scanner, as shown in Figure 2a. Use the L-shape position limit to ensure an optimum and repeatable imaging location. 
	NOTE: It is possible to image two well plates in one scan.</li>
	<li>Scan the plate, as described in step 2.2.</li>
	<li>Run the &#34;Wellplate Reader&#34; software to calculate the required viral titers (Figure 3, step 2.3).
	<ol>
		<li>Click the &#34;Load image&#34; button to load an image. Slide the red bar in &#34;Global Threshold&#34; to adjust the sampling threshold. Click the &#34;Update&#34; button to examine the effect.</li>
		<li>Tick the &#34;Calculate Neutralization/Titration&#34; box. Click the &#34;Sampling&#34; button to quantify the ICP. Click the &#34;Save&#34; button to save the sampling results when prompted. 
		NOTE: After the sampling process, a new window called &#34;Neutralization &#38; Titration Calculation&#34; appears automatically if the &#34;Calculate Neutralization/Titration&#34; box is ticked.</li>
		<li>Load or input the well-plate map. Indicate the threshold (e.g., 50%) in &#34;Neutralization: Infection Deduction (%).&#34;</li>
		<li>Select &#34;Neutralization Process&#34; to calculate the titers. Check the titers and click the &#34;Save &#38; Close&#34; button to save the neutralization results. 
		NOTE: Neutralization is reported as the reciprocal of the highest dilution of the serum with the predefined ICP reduction (such as 50% or 80%) against the VC (the mean of Column 11 in Figure 4). A 50% ICP reduction was used in the Representative Results.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Walker, D. L., Horsfall, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+identity+in+neutralizing+and+hemagglutination-inhibiting+antibodies+against+influenza+viruses.">Lack of identity in neutralizing and hemagglutination-inhibiting antibodies against influenza viruses.</a> J Exp Med. 91, 65-86 (1950).</li><li>Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J. C., van der Werf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemagglutinin+residues+of+recent+human+A(H3N2)+influenza+viruses+that+contribute+to+the+inability+to+agglutinate+chicken+erythrocytes.">Hemagglutinin residues of recent human A(H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes.</a> Virology. 289, 74-85 (2001).</li><li>Lin, Y. P., 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=Neuraminidase+receptor+binding+variants+of+human+influenza+A(H3N2)+viruses+resulting+from+substitution+of+aspartic+acid+151+in+the+catalytic+site:+a+role+in+virus+attachment.">Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment.</a> J Virol. 84, 6769-6781 (2010).</li><li>Harmon, M. W., Rota, P. A., Walls, H. H., Kendal, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+response+in+humans+to+influenza+virus+type+B+host-cell-derived+variants+after+vaccination+with+standard+(egg-derived)+vaccine+or+natural+infection.">Antibody response in humans to influenza virus type B host-cell-derived variants after vaccination with standard (egg-derived) vaccine or natural infection.</a> J Clin Microbiol. 26, 333-337 (1988).</li><li>Rowe, T., Abernathy, R. A., Hu-Primmer, J., Thompson, W. W., Lu, X., Lim, W., 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=Detection+of+antibody+to+avian+influenza+A+(H5N1)+virus+in+human+serum+by+using+a+combination+of+serologic+assays.">Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays.</a> J Clin Microbiol.37. 37, 937-943 (1999).</li><li>Okuno, Y., Tanaka, K., Baba, K., Maeda, A., Kunita, N., Ueda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+focus+reduction+neutralization+test+of+influenza+A+and+B+viruses+in+microtiter+system.">Rapid focus reduction neutralization test of influenza A and B viruses in microtiter system.</a> J Clin Microbiol. 28, 1308-1313 (1990).</li><li>Bachmann, M. F., Ecabert, B., Kopf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus:+a+novel+method+to+assess+viral+and+neutralizing+antibody+titers+in+vitro.">Influenza virus: a novel method to assess viral and neutralizing antibody titers in vitro.</a> J Immunol Methods. 225, 105-111 (1999).</li><li>Matrosovich, M., Matrosovich, T., Garten, W., Klenk, H. 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The authors have nothing to disclose.

In this study, we described an imaging-based MN assay to quantify the true antigenic relationships between viruses. Compared with other plaque-reduction assays, the method emphasizes measuring the ICP of an entire well, ensuring the complete coverage of the infective population. Its independence of plaque formation also widens the application of the assay to viruses that can form mainly invisible small plaques or that can only manage single-cell infection. Therefore, the assay is capable of examining more viruses and the effects of a wider range of antibodies than HI, and it helps to more comprehensively reflect the antigenic similarities or differences between viruses12. For example, when oseltamivir carboxylate is included, the MN assay is less affected by NA-dependent binding, reflecting the antigenic differences more accurately. During the development of the assay, great efforts were made to enhance the experiment consistency, speed, and detection sensitivity, including by controlling input viruses through quantitated titration, imaging in high throughput with a flatbed scanner, and implementing a user-friendly data processing platform. Results have generally been consistent with and confirmed those of HI. Notably, the results also revealed antigenic differences between antigenic drift variants of recent type A and B vaccine viruses, as well as antigenic changes caused by culture-selected or sporadic changes, such as the G155E substitution in HA1 of certain A(H1N1)pdm09 viruses12.
The protocol matched the virus type or subtype with the selection of cell lines that gave the strongest infection, which greatly enhanced the imaging signal. Experimental variation was smoothed with the design of duplicates that balanced with the number of tested antisera. Uncertainties can also come from the image quality, which is mainly influenced by the imaging device. A decent color flatbed scanner can significantly improve the image contrast and reduce the noise. The amount of input virus in the neutralization must be quantitatively determined in advance from the corresponding titration while the viruses still maintain a similar status. During neutralization, the antiserum response to an infection is normalized against the difference between the reference virus (VC) and the background level (CC). Accurate measurements of VC and CC are essential to a neutralization experiment. More duplicates are recommended (eight duplicates were used in the Representative Results). Finally, understanding the software is vital to the success of an experiment. The software has been tested for routine virus characterization in the WHO Influenza Centre, UK for more than two years. Detailed instructions for dealing with various situations is provided in the Supplementary S1.
This MN assay is suitable for applications where the samples cover an extremely large field of view but only require moderate imaging resolution. The low cost and simple setup make the system readily available to most virology laboratories. The variation in measurements of the same sample was less than 3%, which roughly defines the uncertainty introduced during the scanning11. Such reproducibility is sufficient in many virological studies and can be further reduced by averaging images from multiple scans.
In conclusion, this study demonstrates a robust MN assay that can be routinely used in antigenic study and to support HI data in detailed analyses of currently-circulating influenza viruses, notably for the biannual selection of viruses for inclusion in human influenza vaccines.

Micro-neutralization (MN) assays are used in virology for the quantitation of neutralizing antibodies and of antiviral activities. As an alternative of hemagglutination inhibition (HI) assays, MN assays can overcome non-antigenic effects influenced by the affinity changes of receptor-binding in influenza viruses, which can complicate the interpretation of HI results1,2,3. Until recently, most MN assays were based on cytopathic effects (CPE) or enzyme-linked immunosorbent assays (ELISA)4,5. MN assays based on focus and plaque reduction were developed in 19906,7,8. Plaque reduction assays rely on counting visible plaques to quantify infectivity. However, visual counts only cover large plaques that are resolvable by human eyes, even though the majority of plaques are small and invisible for many current circulating viruses. This incomplete coverage can cause significant variation among examiners and between experiments, leading to incomparable results. It is also impossible to use the method when some viruses show abortive infection of single cells or very small plaques.
The poor counting resolution can be improved by the introduction of an imaging-based protocol. With advances in technology, optical microscopy and high-throughput well-plate readers can provide accurate means to count the infected cells9. Under a trans-illuminated microscope, infected cells tagged with certain markers may be visualized by their absorption or fluorescence contrast in sub-cellular resolution. A sample can then be analyzed on a computer screen.
Unfortunately, due to the limitation in the field of view, more than a hundred tiled images are required to cover a single well. Analyzing a plate with 96 wells would require the imaging and processing of about ten thousand images. Such a laborious process is time consuming and expensive, and the resolution gained is in general unnecessary for routine characterization of viral infections. Laboratories with a limited budget may find that the approach built around a flatbed scanner provides a cost-effective, high-throughput alternative.
In this paper, we describe an improved plaque reduction MN assay that is suitable for the antigenic characterization of a large number of viruses and for quantitatively measuring antiviral activities and neutralization antibodies. The assay has several advantages: firstly, it is an imaging-based assay that is able to measure virus infections on the cellular level, regardless of plaque size. Counting the total infected cell population (ICP) within a well greatly increases the detection sensitivity, making it possible to characterize the viruses with low infectivity. Secondly, a more accurate quantitative titration is introduced prior to neutralization to determine the amount of input virus. The quantitative input virus significantly reduces the variation between different experiments and makes the results more comparable between laboratories. Thirdly, neutralization titers can be determined directly by analyzing images, making the quantitation fast and user-friendly. Finally, the protocol provides a cost-effective and high-throughput alternative with the required resolution and accuracy. The quantitation is based on a flatbed scanner and free data processing software. The whole setup has a small footprint and is deployable in most laboratories.
The protocol presented in this paper consists of four major steps, including virus titration, titration quantitation, virus neutralization, and neutralization quantitation. Virus titration is a preparation experiment that determines the amount of input viruses to be used in neutralization. During a titration, a number of viral concentrations are applied to the cell monolayers in a 96-well plate. The infected cells are then quantitated in section 2. The viral dilution that produces 20%-85% ICP is in turn applied as input viruses to the corresponding neutralization in section 3. The titers of the neutralization protocol are quantified using section 4. Experiments that followed the above protocols are presented in the Representative Results. The assay has been tested thoroughly during the last two years with most of the current circulating influenza viruses, such as A(H1N1)pdm09, A(H3N2), and B viruses. The results of the influenza virus characterization were included in the reports for the WHO consultation meeting that gave recommendations on influenza vaccines for use in the Southern Hemisphere in 2016 and the Northern Hemisphere in 2016-2017.

<table border="0" cellpadding="0" cellspacing="0" width="1115">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">VGM</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">D6429, P0781</td>
			<td style="width:383px;">500 ml DMEM+5 ml Pen/Strepb</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:177px;">PBS A</td>
			<td style="width:172px;"></td>
			<td style="width:383px;"></td>
			<td style="width:383px;">Nature pH Phosphate-buffered saline: NaCl -10 gm, KCl - 0.25 gm, Na2HPO4 - 1.437 gm, KH2PO4 &#8211; 0.25 .gm, and Dist. Water &#8211; 1 L.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:177px;">Avicell</td>
			<td style="width:172px;">FMC</td>
			<td style="width:383px;">RC-581F</td>
			<td style="width:383px;">2.4g in 100ml distilled water dissolved by agitation on a magnetic stirrer for 1 hr. Sterilize by autoclaving.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">2XDMEM</td>
			<td style="width:172px;">Gibco&#160;</td>
			<td style="width:383px;">21935-028</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Trypsin</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">T1426</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:177px;">Overlay (10ml/plate):</td>
			<td style="width:172px;"></td>
			<td style="width:383px;"></td>
			<td style="width:383px;">5ml 2X DMEM, Trypsin 2&#956;g/ml final conccentration,Avicell&#160; - 5ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Triton X- 100e</td>
			<td style="width:172px;">Sigma&#160;</td>
			<td style="width:383px;">T8787</td>
			<td style="width:383px;">Permeabilisation buffer: 0.2% in PBS A (v/v)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Tween 80</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">P5188</td>
			<td style="width:383px;">&#160;Wash Buffer: 0.05% Tween &#8211; 80 in PBS A (v/v)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:177px;">Horse serum</td>
			<td style="width:172px;">PAA Labs Ltd</td>
			<td style="width:383px;">B15-021</td>
			<td style="width:383px;">ELISA Buffer: 10%&#160; in PBS A (v/v) + 0.1% Tween 80</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">Mouse MAb against influenza type A</td>
			<td style="width:172px;">Biorad</td>
			<td style="width:383px;">MCA 400</td>
			<td style="width:383px;">1st Antibody: 1:1000 in ELISA Buffer</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">Goat anti-mouse IgG (H+L) HRP conjugate</td>
			<td style="width:172px;">Biorad</td>
			<td style="width:383px;">172-1011</td>
			<td style="width:383px;">2nd Antibody: 1:1000 in ELISA Buffer</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">True Blu peroxidase substrate</td>
			<td style="width:172px;">KPL&#160;</td>
			<td style="width:383px;">50-78-02</td>
			<td style="width:383px;">Substrate:&#160; True blue +0.03% H2O2g (1:1000 of 30% solution)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">96-well flat-bottom microtitre plates</td>
			<td style="width:172px;">Costar&#160;</td>
			<td style="width:383px;">3596</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:177px;">8 channel multiwall-plate washer and manifold</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:383px;">M2656</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="104">
			<td height="104" style="height:104px;width:177px;">Perfection Plate Scanner</td>
			<td style="width:172px;">Epson</td>
			<td style="width:383px;">V750 Pro</td>
			<td style="width:383px;"><a href="http://www.epson.com/cgi-bin/Store/support/supDetail.jsp?oid=66134&amp;infoType=Downloads.">The imaging software can be downloaded freely from http://www.epson.com/cgi-bin/Store/support/supDetail.jsp?oid=66134&#38;infoType=Downloads.</a></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:177px;">Software operational environment: LabVIEW</td>
			<td style="width:172px;">National Instruments Corporation</td>
			<td style="width:383px;">Window based with NI Vision builder</td>
			<td style="width:383px;">Version: LabVIEW2012 or above</td>
		</tr>
	</tbody>
</table>

optimization of a quantitative micro-neutralization assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

Following the above procedures, we present some results from recent antigenic characterization experiments. The titration setup was designed to examine eight input viruses, with double duplicates for each dilution. The viral dilutions were tested between 1.0 x 10-1 and 1.0 x 1.0-6 to cover the variations between the viruses. The test viruses were from the H3N2 subtype, including A/Cameroon/15V-3538/2015, A/Vladivostok/36/2015, A/Moscow/103/2015, A/Tomsk/5/2015/, A/Moscow/101/2015, A/Moscow/100/2015, A/Moscow/133/2015, and A/Bratislava/437/2015. The titration results are illustrated in Figure 5. Figure 5d demonstrates the decrease of ICPs with the increase of virus dilution. The curves were normalized against the ICPs of the same viruses that yielded infections in all cells within a well12 (defined as ICP saturation). If the ICP did not reach saturation with the highest virus concentration, the ICP average from corresponding duplicates was used instead (A/Moscow/103/2015 in Figure 5d). The virus dilutions that produced 30% of ICP saturation were chosen as the input virus dilution for neutralization (Table 3).
Neutralization aimed to have one input virus against five antisera, with double duplicates on each plate. The reference virus shown is H3N2 A/Stockholm/63/2015. The five antisera are A/HK 4801/14 Egg F12/15, A/HK 7295/14 MDCK F02/15, A/South Africa R2665/15 SIAT F50/15, A/Swiss 9715923/13 SIAT NIBF F18/15, and A/Dutch 525/14 SIAT f23/15. The neutralization results are illustrated in Figure 6. Figure 6d shows the infection progress with the increase of serum dilution. The normalized positive population on the vertical axis represents the ratio of ICPs from the corresponding antisera response against the average ICP of the reference virus12. The background ICP from uninfected cell controls was subtracted during the normalization. The neutralization titers were determined as the reciprocals of the antiserum dilutions corresponding to 50% ICP reduction (Table 4). Linear interpolation was used to estimate titers falling between two adjacent serum dilutions.
<img alt="Figure 1" src="/files/ftp_upload/54897/54897fig1.jpg" /> 
Figure 1: Example of a well-plate setup in a virus titration experiment. Test viruses are assigned to separate rows (A to H). Columns are designed for different viral dilutions (1 to 12). Two columns were used as duplicates for each viral dilution. Scale bar = 10 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54897/54897fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54897/54897fig2.jpg" /> 
Figure 2: Schematics of a sample scanning system. (a) A 96-well plate on the imaging position of a flatbed scanner (republished from reference11 with permission from Elsevier B.V.), and (b) dimensions of the L-shape position limit shown in (a). <a href="http://cloudflare.jove.com/files/ftp_upload/54897/54897fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54897/54897fig3.jpg" /> 
Figure 3: Desktop diagram of the &#34;Wellplate Reader&#34; quantitation software. <a href="http://cloudflare.jove.com/files/ftp_upload/54897/54897fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54897/54897fig4.jpg" /> 
Figure 4: Example of a well-plate setup in a neutralization experiment. Each antiserum is assigned to two columns with duplicates (1 to 10). Rows are designed for different serum dilutions (A to H). The virus control takes Column 11, with eight duplicates (A11 to H11). The cell control is in Column 12, with eight duplicates (A12 to H12). Scale bar = 10 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54897/54897fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54897/54897fig5.jpg" /> 
Figure 5: Results of a titration experiment. (a) The well-plate setup, (b) scanned well-plate image, (c) illustration of the color-encoded, quantitated, virus-infected cell population, and (d) normalized virus-infected cell populations against virus dilutions. 30% ICP was used as the threshold. The error bars in (d) are standard deviations (SD) from the sample duplications (&#177; 1 SD). Scale bars in (b) and (c) = 10 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54897/54897fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54897/54897fig6.jpg" /> 
Figure 6: Results of a neutralization experiment. (a) The well-plate setup, (b) scanned well-plate image, (c) illustration of the quantitated, virus-infected cell population, and (d) normalized virus-infected cell population against the serum dilution. The input virus (VC) is A/Stockholm/63/2015. 50% ICP was used to calculate the titers. The error bars in (d) are standard deviations (SD) from the sample duplications (&#177; 1 SD). Scale bars in (b) and (c) = 10 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54897/54897fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary S1: <a href="http://cloudflare.jove.com/files/ftp_upload/54897/Supplementary_S1.pdf" target="_blank">Please click here to download this file.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Typical dilution</td>
		</tr>
		<tr>
			<td></td>
			<td>MDCK cells</td>
			<td>MDCK-SIAT1 cells</td>
		</tr>
		<tr>
			<td>2 days</td>
			<td>1:5 (1 + 4)</td>
			<td>1:10 (1 + 9)</td>
		</tr>
		<tr>
			<td>3 days</td>
			<td>1:10 (1 + 9)</td>
			<td>1:20 (1 + 19)</td>
		</tr>
		<tr>
			<td colspan="3">Typical number of cells per ml</td>
		</tr>
		<tr>
			<td></td>
			<td>MDCK cells</td>
			<td>MDCK-SIAT1 cells</td>
		</tr>
		<tr>
			<td>2 days</td>
			<td>2 x 105</td>
			<td>1 x 105</td>
		</tr>
		<tr>
			<td>3 days</td>
			<td>1 x 105</td>
			<td>5 x 104</td>
		</tr>
	</tbody>
</table>
Table 1: Typical dilutions on a subculture of confluent flasks of MDCK or MDCK-SIAT1 cells.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mode</td>
			<td>Professional Mode</td>
		</tr>
		<tr>
			<td>Document Type</td>
			<td>Film (with Film Area Guide)</td>
		</tr>
		<tr>
			<td>Auto Exposure Type</td>
			<td>Photo</td>
		</tr>
		<tr>
			<td>Image Type</td>
			<td>24-bit Color</td>
		</tr>
		<tr>
			<td>Resolution</td>
			<td>1,200 dpi</td>
		</tr>
		<tr>
			<td>Saved image Format</td>
			<td>TIFF</td>
		</tr>
	</tbody>
</table>
Table 2: Typical setup of a flatbed scanner.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Row</td>
			<td>Virus</td>
			<td>Recommended dilution</td>
		</tr>
		<tr>
			<td>A</td>
			<td>A/Cameron/15V-3538/2016</td>
			<td>1.0 x 10-2</td>
		</tr>
		<tr>
			<td>B</td>
			<td>A/Vladivostok/36/2015</td>
			<td>1.0 x 10-3</td>
		</tr>
		<tr>
			<td>C</td>
			<td>A/Moscow/103/2015</td>
			<td>1.1 x 10-1</td>
		</tr>
		<tr>
			<td>D</td>
			<td>A/Tomsk/5/2015</td>
			<td>5.9 x 10-1</td>
		</tr>
		<tr>
			<td>E</td>
			<td>A/Moscow/101/2015</td>
			<td>8.3 x 10-1</td>
		</tr>
		<tr>
			<td>F</td>
			<td>A/Moscow/100/2015</td>
			<td>1.4 x 10-2</td>
		</tr>
		<tr>
			<td>G</td>
			<td>A/Moscow/133/2015</td>
			<td>1.3 x 10-2</td>
		</tr>
		<tr>
			<td>H</td>
			<td>A/Bratislava/437/2015</td>
			<td>1.3 x 10-4</td>
		</tr>
	</tbody>
</table>
Table 3: Viral dilutions calculated from a population of 30% ICP.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Column</td>
			<td>Antisera</td>
			<td>Recommended titer</td>
		</tr>
		<tr>
			<td>1 - 2</td>
			<td>A/HK4801/2014</td>
			<td>110</td>
		</tr>
		<tr>
			<td>3 - 4</td>
			<td>A/HK7295/2014</td>
			<td>213.3</td>
		</tr>
		<tr>
			<td>5 - 6</td>
			<td>A/South Africa/R2665/2015</td>
			<td>2155.8</td>
		</tr>
		<tr>
			<td>7 - 8</td>
			<td>A/Switzerland/9715293/2013</td>
			<td>89.4</td>
		</tr>
		<tr>
			<td>9 - 10</td>
			<td>A/Netherlands/525/2014</td>
			<td>78.6</td>
		</tr>
	</tbody>
</table>
Table 4: Titers at 50% ICP of A/Stockholm/63/2015 against antisera.

Watch this Scientific Journal Video about Optimization of a Quantitative Micro-neutralization Assay at JoVE.com

Optimization of a Quantitative Micro-neutralization Assay

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

optimization-of-a-quantitative-micro-neutralization-assay

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

20.9K Views.  The Francis Crick Institute. This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

Video: Optimization of a Quantitative Micro-neutralization Assay

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

We developed an in vitro model that mimics an important component of the acute inflammatory response during infection of the bladder with uropathogenic Escherichia coli. The transuroepithelial neutrophil migration assay enables quantitative assessment of human neutrophil migration across bladder epithelia, cultured on permeable supports, in response to bacterial infection or chemoattractant substances.

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

Optimization of a Quantitative Micro-neutralization Assay

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Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria