We thank members of the Wobus laboratory for critical comments and suggestions. Work in the laboratory of CEW was funded by start-up funds from the University of Michigan, a career development grant from the NIH/NIAID Regional Center of Excellence for Bio-defense and Emerging Infectious Diseases Research (RCE) Program, Region V 'Great Lakes' RCE (NIH award 1-U54-AI-057153) and NIH R01 AI080611. M.B.G.-H. was funded by the Experimental Immunology (NIH T32 A1007413-16) and the Molecular Mechanisms in Microbial Pathogenesis (NIH T32 A1007528) training grants to the University of Michigan. J.B.C. was funded by Coordena&ccedil;&atilde;o de Aperfei&ccedil;oamento de Pessoal de N&iacute;vel Superior (CAPES), Brasilia, Brazil.

1. Culturing of the Macrophage Cell Line RAW 264.7
<ol>
	<li>Maintain RAW 264.7 cells (ATCC, catalog # TIB-71) in DMEM-10 media, which consists of high glucose DMEM with 10% (v/v) low-endotoxin fetal bovine serum (&lt; 10 EU/ml), 10 mM HEPES, 100 U/ml penicillin, 100 &mu;g/ml streptomycin, 1 mM non-essential amino acids, 2 mM L-glutamine. Cells are typically maintained in 175 cm2 tissue culture flasks containing 35 ml of media per flask and incubated at 37 &deg;C and 5% CO2 in a tissue culture incubator. However, any size flask can be used with a volume of media that is appropriate for the size of the flask.</li>
	<li>To split cells: aspirate off the old media, add 10 ml of fresh DMEM-10 media to the cells, and then scrape the cells from the bottom of the flask by using a cell scraper. Next, resuspend the cells into a homogenous solution by drawing up cells into a 10 ml pipette and forcefully squeezing the cells through the pipette tip pressed against the bottom of the flask. Repeat this action at least 3 times so the cells no longer clump together. Verify by light microscopy that a single cell suspension was generated. Then transfer 1 ml (1:10 dilution or ~1x107 cells) - 2 ml (1:5 dilution or ~2x107 cells) of the cell suspension to a new 175 cm2 flask, and bring the final volume of media up to 35 ml.</li>
	<li>Split cells when they are nearly confluent (~1x108 cells total/175 cm2 flasks): every three days if starting with a 1:10 dilution, or every two days if starting with a 1:5 dilution. Use light microscopy to check cell morphology before splitting cells. Most of the cells should look round and not activated. Activated cells have granules and/or extended, spindly morphology with appendages. Do not let cells overgrow as those cells do not typically form plaques. Keep track of the passage number and frequently start over by thawing a lower passage aliquot of cells. (We use passage 30 as a cut-off).</li>
</ol>
2. Infect RAW 264.7 Cells with MNV Inoculum
<ol>
	<li>Seed RAW 264.7 cells into 6-well plates (3.5 cm diameter) at a density of 1x106 viable cells/ml in DMEM-10 media, and add 2 ml of this suspension to each well. It is important to distribute cells evenly in wells either by rocking plates by hand at least 10 times or by using a rocking apparatus for ~10 min. Do not swirl the plates as this will cause the cells to cluster in the center of the well. Place plates into a tissue culture incubator (at 37 &deg;C and 5% CO2). Allow cells to attach overnight or for at least 4 hr at 37 &deg;C. Cells should be 60 - 80% confluent for the plaque assay and distributed evenly throughout the well.</li>
	<li>The next day, prepare the virus inoculum, which can be from MNV-infected cells in tissue culture or from homogenized tissues or fecal samples of MNV-infected mice. When using tissue samples, pea-sized pieces of tissue are homogenized in 2 ml screw-cap tubes containing sterile silica beads in 1 ml of DMEM-10 using a tissue homogenizer (e.g. MagnaLyser; Roche). For fecal samples, no more than 3 fecal pellets should be homogenized in 1 ml media. All samples are then frozen (at -80 &deg;C) and thawed once before performing the plaque assay.</li>
	<li>Prepare 10-fold dilutions of the virus inoculum in complete DMEM-5 medium, which consists of DMEM/High glucose, 5% (v/v) low-endotoxin fetal bovine serum (&lt; 10 EU/ml), 10 mM HEPES, 100 U/ml penicillin, 100 &mu;g/ml streptomycin, 1 mM non-essential amino acids, 2 mM L-glutamine.</li>
	<li>Ten-fold serial dilutions are prepared in 24-well plates: A repeater pipette is used to dispense 1.35 ml media into multiple wells, the 10-1 dilution is made by mixing 1.35 ml of media and 0.15 ml of virus-containing sample, and then 0.15 ml of the 10-1 dilution is added to 1.35 ml of media to make the 10-2 dilution and so on. It is important to change tips each time you make a new dilution. A multichannel pipette can be used to make the dilutions of multiple samples at a time with two tips fitting into one well of a 24-well plate transferring a total volume of 0.15 ml per well (see Figure 3A).</li>
	<li>A typical dilution range for tissue homogenates and fecal contents is 10-1 to 10-3. However, plaques from these samples tend to be smaller compared to those from tissue culture samples. Furthermore, in some cases a 1:100 dilution of fecal samples is needed to sufficiently dilute out any toxic components of the feces that may disrupt the cell monolayer, thus hindering the ability to count plaques. The dilution range of tissue culture lysates depends on the time point of interest during the viral life cycle. Dilutions that go up to 10-9 may be needed at the peak of infection.</li>
	<li>After the serial dilutions are prepared, label the 6-well plates containing RAW 264.7 monolayers (from section 2.1) with the sample name and dilutions being plated. One plate at a time, remove all media by flicking it out or aspirating it. Immediately afterwards add 0.5 ml of a diluted sample to a well, then repeat with a duplicate well, before proceeding to the next dilution. Once all 3 dilutions are added to one plate, tilt plate back and forth by hand to ensure all cells have been covered. Handle one plate at a time to ensure that cells will not dry out.</li>
	<li>After adding 0.5 ml of the dilutions to each well, stack plates upright and incubate them for 1 hr at room temperature. Because the volume added to each well is not sufficient to cover the monolayer completely, the plates need to be gently tilted back and forth by hand every 10-15 min or placed on a rocking apparatus (~18 oscillations per min). This prevents cells from drying out.</li>
</ol>
3. Low Melting Point Agarose (SeaPlaque) Overlay Preparation
Note: it is advisable to have several bottles with autoclaved SeaPlaque agarose prepared ahead of time. Agarose can be re-melted in a microwave before use.
<ol>
	<li>Calculate the amount of overlay required for the total volume of plates before the 1 hr incubation is complete. The volume needed is 2 ml/well or 12 ml/6-well plate. Prepare agarose (see section 3.2) and media (see section 3.3) separately.</li>
	<li>To prepare the agarose, suspend 3 g of SeaPlaque agarose in a total volume of 100 ml of distilled water (3% w/v) in a glass bottle. Autoclave for 20-30 min. (If agarose was already prepared before-hand, re-melt agarose in microwave.) It is important to equilibrate SeaPlaque agarose to 42 &deg;C in a water bath before use because if the agarose is too hot, it will kill the cells. Make sure water level is equal to or above the level of the agarose to avoid undesired solidification.</li>
	<li>To prepare the media: make 100 ml of 2x MEM media, which consists of 2x MEM, 10% (v/v) low-endotoxin fetal bovine serum (&lt; 10 EU/ml), 10 mM HEPES, 100 U/ml penicillin, 100 &mu;g/ml streptomycin, 4 mM L-glutamine. Equilibrate media to 37 &deg;C in a water bath.</li>
	<li>Mix both the SeaPlaque agarose and the 2x MEM media together in a sterile bottle at a 1:1 ratio immediately before overlaying the infected cell monolayers. If more than 200 ml overlay is needed, split volume into multiple bottles and keep in 37 &deg;C water bath until ready to use.</li>
	<li>At the end of the 1 hr incubation (see section 2.7), aspirate the inoculum off of each well. Slowly add 2 ml of overlay to the edge of each well by placing the pipette tip against the wall of each well. Up to 5 plates can be handled simultaneously without cells drying out.</li>
	<li>Allow the overlay to solidify for approximately 10 min at room temperature before placing plates upright into the tissue culture incubator. Incubate plates for 48 hr at 37 &deg;C in 5% CO2.</li>
	<li>After the incubation period, plaques are faintly visible to the naked eye, so check unstained plates for presence of plaques. If no plaques are visible, incubate for an additional 4 hr and check again. However, the maximum incubation time should not exceed 72 hr.</li>
</ol>
4. Visualization of Plaques by Neutral Red Staining
<ol>
	<li>To visualize plaques, the neutral red staining solution is prepared by adding 3 ml of neutral red (0.33% w/v in DPBS; Sigma, catalog # N2889) to every 97 ml of 1x PBS (tissue culture grade, Mg2+-, Ca2+-free; Gibco, catalog # 10010). Calculate the volume of neutral red staining solution needed for the experiment: 12 ml neutral red staining solution are required for each 6-well plate. Then, add 2 ml to each well. Although some plaque assay protocols require the agarose plug to be removed from the wells, in this protocol the neutral red staining solution is added directly onto the overlay.</li>
	<li>After a one hr incubation at 37 &deg;C, check if plaques are visible with neutral red staining solution still in wells. If plaques are not readily apparent, allow the staining to continue for another hr. Continue incubating until plaques are visible. (Note: Staining for more than 3 hr is not optimal and if no plaques are visible in the positive control sample after 3 hr of staining, the plaque assay did not work properly.) After the staining is complete, aspirate the neutral red staining solution, ensuring the agarose plug is not disturbed, and then proceed to counting the plaques.</li>
	<li>Count plaques by placing plate upside down on a light box and marking a dot on counted plaques to avoid duplicate counts. Choose the dilution to count plaques in wells where plaques are clearly separated (i.e. no visual evidence of plaques fusing together). If possible, count plaques at two dilutions. It is important to note that plaque size may vary between MNV strains, virus inoculum, and depends on the condition of the RAW 264.7 cells during the plaque assay.</li>
	<li>If no plaques are visible in a well, either there was no virus present in the sample or the amount of virus was under the limit of detection of the plaque assay. In this case, the wells stain red with a similar color as other plaque-containing wells. Alternatively, the absence of plaques is also observed when there are too many viral particles present in a given dilution. This leads to lysis of the entire monolayer and wells appear orange/yellow in color.</li>
	<li>Calculate viral titers. Add the number of plaques in both wells at a single dilution and multiply by the dilution factor (i.e. 1 ml if 2 wells are infected with 0.5 ml). This will yield the amount of plaque forming units (PFU) in your inoculum volume of 1 ml. For example, in Figure 4 at the 10-2 dilution, one well (marked "II") has 14 plaques and the other well (marked "V") has 17 plaques. Thus, the viral titer will be 14x102 + 17x102 = 3,100 (3.1x103) pfu/ml.</li>
</ol>
5. Representative Results
Infectious MNV-1 particles can be quantified using a plaque assay as outlined schematically in Figure 1. Figure 2A shows a well with a monolayer of RAW 264.7 cells just prior to infection, while Figure 2B shows three visible plaques indicated by roman numbers I, II and III in a well. Individual steps of the assay are depicted in Figures 3A through F. Figure 3A shows the preparation of the 10-fold dilution series of a virus-containing sample. Figure 3B shows the transfer of dilutions to duplicate wells of a 6-well plate. Figure 3C shows the rocking apparatus used to incubate RAW 264.7 cells with the inoculum at room temperature for 1 hr. Figure 3D shows cells being overlaid with the SeaPlaque:MEM mixture. Figure 3E shows a plate at room temperature to allow the overlay to solidify, while Figure 3F shows cells being stained with a 0.01% neutral red solution 48 hr later. After staining cells for 1-3 hr and aspirating the neutral red staining solution, plaques are visible and can be counted (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/4297/4297fig1.jpg" /> 
 Figure 1. Schematic of the MNV plaque assay protocol.
<img alt="Figure 2" src="/files/ftp_upload/4297/4297fig2.jpg" /> 
 Figure 2. Representative images of a well of a monolayer before infection and after formation of plaques. A) RAW 264.7 cells were cultured overnight and imaged under a light microscope at 20x magnification. B) Cells were stained with a 0.01% neutral red solution after 48 hr of infection and visualized under a light microscope at 4x magnification. Roman numbers I, II, and III indicate three visible plaques.
<img alt="Figure 3" src="/files/ftp_upload/4297/4297fig3.jpg" /> 
 Figure 3. Representative images of the different plaque assay steps. A) MNV-1 inoculum is prepared in 10-fold dilutions. B) Inoculum is added to cell monolayers in duplicate wells. C) Cells and inoculum are incubated by rocking for 1 hr at room temperature. D) Cells are overlaid with a 1:1 mixture of SeaPlaque agarose and 2x MEM media. E) Plates are incubated for 10 min at room temperature to allow the overlay to solidify. F) Staining of cells with the neutral red staining solution 48 hr post-infection.
<img alt="Figure 4" src="/files/ftp_upload/4297/4297fig4.jpg" /> 
 Figure 4. MNV-1 forms plaques in cell monolayers. Shown here is a representative plaque assay plate 48 hr post-infection, showing plaques stained with neutral red staining solution after 1 hr of incubation. The plate shows duplicate wells of three 10-fold dilutions. Wells labeled with roman numbers I and IV correspond to the 10-1 dilution; II and V correspond to the 10-2 dilution; III and VI correspond to the 10-3 dilution. The viral titer of the sample is indicated below (see Section 4.5 for details of the calculation).

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	<li>Wobus, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+Norovirus+in+cell+culture+reveals+a+tropism+for+dendritic+cells+and+macrophages.">Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.</a> PLoS Biol. 2, e432 (2004).</li><li>Wobus, C. E., Thackray, L. B., Virgin, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+norovirus:+a+model+system+to+study+norovirus+biology+and+pathogenesis.">Murine norovirus: a model system to study norovirus biology and pathogenesis.</a> Journal of virology. 80, 5104-5112 (2006).</li><li>Condit, R. C., Knipe, D. M., Howley, 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=Ch.+2.">Ch. 2.</a> Fields Virology. 1, 25-58 (2007).</li><li>Thackray, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+noroviruses+comprising+a+single+genogroup+exhibit+biological+diversity+despite+limited+sequence+divergence.">Murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence.</a> Journal of virology. 81, 10460-10473 (2007).</li><li>Reed, L. J., Muench, 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+simple+method+for+estimating+50%+endpoints.">A simple method for estimating 50% endpoints.</a> American Journal of Hygiene. 27, 493-497 (1932).</li><li>Chachu, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+is+critical+for+the+clearance+of+murine+norovirus+infection.">Antibody is critical for the clearance of murine norovirus infection.</a> Journal of virology. 82, 6610-6617 (2008).</li><li>Barron, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+murine+norovirus+strains+isolated+from+asymptomatic+mice+of+different+genetic+backgrounds+within+a+single+U.S.+research+institute.">Diversity of murine norovirus strains isolated from asymptomatic mice of different genetic backgrounds within a single U.S. research institute.</a> PLoS ONE. 6, e21435 (2011).</li><li>Cox, C., Cao, S., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+detection+and+study+of+murine+norovirus-1+using+a+more+efficient+microglial+cell+line.">Enhanced detection and study of murine norovirus-1 using a more efficient microglial cell line.</a> Virology journal. 6, 196 (2009).</li><li>Cooper, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plaque+assay+of+animal+viruses.">The plaque assay of animal viruses.</a> Adv. Virus Res. 8, 319-378 (1961).</li><li>Hyde, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+norovirus+replication+is+associated+with+virus-induced+vesicle+clusters+originating+from+membranes+derived+from+the+secretory+pathway.">Mouse norovirus replication is associated with virus-induced vesicle clusters originating from membranes derived from the secretory pathway.</a> Journal of virology. 83, 9709-9719 (2009).</li><li>Simmonds, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinformatic+and+functional+analysis+of+RNA+secondary+structure+elements+among+different+genera+of+human+and+animal+caliciviruses.">Bioinformatic and functional analysis of RNA secondary structure elements among different genera of human and animal caliciviruses.</a> Nucleic acids research. 36, 2530-2546 (2008).</li></ol>

No conflicts of interest declared.

The plaque assay method for MNV-1 presented here is a way of quantifying infectious MNV particles. By following the assay steps illustrated in Figure 3, one can obtain reproducible viral titers. The limit of detection of the assay depends on the starting dilution used. When starting with a 1:10 dilution of sample as described above, the limit of detection of the plaque assay is 10 pfu (i.e., 1 plaque visible at the 10-1 dilution). Since each plaque represents a single virus, the plaque assay can also be used to purify clonal populations of MNV by picking isolated plaques and propagating them as described previously 1. In addition, plaque purifications can also be used to separate an individual virus population from mixed virus populations. A limitation of using a plaque assay for the detection of MNV infection is that not all MNV strains form plaques 4. However, it may be possible to overcome the inability of some MNV strains, isolated from animals, to form plaques by serially passaging these viruses in tissue culture 7. An alternative to the plaque assay is to measure infectious particles via the TCID50 technique 3, 4. This assay quantifies the amount of virus required to produce CPE in 50% of inoculated tissue culture cells following endpoint dilutions and takes 1 week to complete for MNV 4. In addition to being slower than a plaque assay, the TCID50 assay is also not as sensitive (limit of detection = 200 TCID50/ml) due to the toxicity of tissue samples to RAW 264.7 cells 4.
Although critical steps within the protocol have been described throughout the protocol, the following section provides a summary to facilitate trouble-shooting. The most critical step in the protocol is to ensure that RAW 264.7 cells remain viable throughout the assay to support virus replication. This can be monitored at each stage of the assay via light microscopy. Cell viability is ensured in two ways. First, care should be taken not to let cells dry out while handling plates. Thus, plates are inoculated one at a time, rocked during the infection period, and should remain closed whenever they are not being handled. Second, solutions added onto cells should be equilibrated to ~37 &deg;C. Furthermore, it is vital for the overall health of the RAW 264.7 cells to maintain them in media containing low endotoxin serum (&lt; 10 EU/ml), which limits activation of cells. In addition, we have observed a higher failure rate of the plaque assay when using cells from passage 30 or higher. Although this will likely vary from lab to lab, it is important to include a positive control (e.g., a sample with a known viral titer) to ensure reproducible titers, especially when using higher passage RAW 264.7 cells. To limit use of higher passage cells, it is advisable to freeze vials of early passage cells upon receipt of RAW 264.7 cells and start a new culture from the frozen vials frequently. Starting over with low passage cell cultures will also be helpful when cells exhibit altered characteristics, such as failure to adhere, changes in cell morphology (e.g. from round to spindly and spread out), or when mycoplasma contamination has been detected. Another important point to pay attention to is to ensure that pipette tips are changed between samples and during dilutions. This will ensure accurate serial dilutions and prevent cross-contamination between samples. The one step in the protocol where the same pipette tip can be used again is when serial dilutions of the same sample are added to wells. In that case, one should start from the most diluted inoculum to the least, and vigorously pipette up and down when drawing up a new dilution.
The plaque assay protocol is amendable to several modifications. One modification that can be made when there are not enough cells for inoculating wells in duplicate is to inoculate only a single well for each dilution. However, since the inoculum volume is 0.5 ml, the number of plaques then needs to be multiplied by a factor of 2 to normalize to pfu/ml. The plaque assay can also be adapted for use with any other adherent cell line that is able to support replication of MNV, and this has been described for the murine microglial BV-2 cell line 8. Other modifications that can be implemented are adaptations that have been described for plaque assay protocols developed for other viruses. In case of MNV, the following modifications have already been implemented successfully; the use of methyl cellulose instead of Sea Plaque agarose 9, and staining of cells with crystal violet or methylene blue instead of neutral red 10, 11.
Overall, this protocol can easily be adapted as needed to quantify other plaque-forming viruses or used for other viruses that cause lytic infections in RAW 264.7 cells, making it a useful tool to quantify infectious viral particles in general.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM/ High glucose</td>
 <td>Hyclone</td>
 <td>SH30243.02</td>
 </tr>
 <tr>
 <td>2x MEM</td>
 <td>Gibco</td>
 <td>11935</td>
 </tr>
 <tr>
 <td>100x Penicillin and streptomycin</td>
 <td>Hyclone</td>
 <td>SV30010</td>
 </tr>
 <tr>
 <td>10 mM Non-essential amino acids</td>
 <td>Hyclone</td>
 <td>SH30238.01</td>
 </tr>
 <tr>
 <td>1M HEPES</td>
 <td>Hyclone</td>
 <td>SH30237.01</td>
 </tr>
 <tr>
 <td>200 mM (100x) L-glutamine</td>
 <td>Hyclone</td>
 <td>SH30034.01</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Gibco, Hyclone</td>
 <td>10437, SH30070.02</td>
 </tr>
 <tr>
 <td>Sea Plaque Agarose</td>
 <td>Lonza</td>
 <td>50100</td>
 </tr>
 <tr>
 <td>Neutral Red 0.33%</td>
 <td>Sigma</td>
 <td>N2889</td>
 </tr>
 <tr>
 <td>1x PBS</td>
 <td>Gibco</td>
 <td>10010</td>
 </tr>
 <tr>
 <td>1.0 mm Zirconia/Silica beads</td>
 <td>BioSpec Products</td>
 <td>11079110z</td>
 </tr>
 <tr>
 <td>Model 35 Speed Rocker</td>
 <td>Labnet</td>
 <td>S2035 </td>
 </tr>
 <tr>
 <td>Magna Lyser Instrument</td>
 <td>Roche</td>
 <td>03358968001 </td>
 </tr>
 <tr>
 <td>Raw 264.7 cell line</td>
 <td>ATCC</td>
 <td>TIB-71</td>
 </tr>
 <tr>
 <td>Tissue culture incubator</td>
 <td>Sanyo</td>
 <td>MCO-18AIC </td>
 </tr>
 </tbody>
</table>

plaque assay for murine norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Watch this Scientific Journal Video about Plaque Assay for Murine Norovirus at JoVE.com

Plaque Assay for Murine Norovirus

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

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JoVE Journal

Immunology and Infection

54.3K ビュー.  University of Michigan, Ann Arbor. ここでは、効率的に細胞培養で複製のみノロウイルスですマウスノロウイルス（MNV）の感染性粒子を定量化する方法を説明します。プラークアッセイは、マウスのマクロファージのためにMNVの向性を活用し、MNVを含む生物学的または環境試料での使用に適合させることができる。

ビデオ: Plaque Assay for Murine Norovirus

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.