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1. VLP-bacteria Attachment Assay
CAUTION: Human norovirus VLPs are a biosafety level (BSL)-2 hazard, and all work involving VLPs should be performed in a biosafety cabinet. Preparation of the bacterial cultures before the attachment assay should be performed using safety conditions appropriate for the organism.
<ol>
	<li>Reagent preparation&#8203;
	<ol>
		<li>1x phosphate-buffered saline (PBS)
		<ol>
			<li>Prepare 1 L of 10x PBS by dissolving a packet in 1 L of deionized (DI) water.</li>
			<li>Autoclave solution for 30 min.</li>
			<li>Prepare a 1x solution by diluting the above solution 1:10 in DI water.</li>
			<li>Filter sterilize the solution by passing it through a 0.22 &#956;m filter and storing it at room temperature.</li>
		</ol>
		</li>
		<li>5% BSA
		<ol>
			<li>Add 5 g of bovine serum albumin (BSA) powder to 100 mL of PBS to generate a 5% (w/v) solution.</li>
			<li>Mix the solution by vortexing or using a metal stir bar on a stir plate until clumps have dissolved.</li>
			<li>Filter sterilize using a 0.22 &#956;m filter.</li>
			<li>Store the solution at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Flow cytometry stain buffer (FCSB)
		<ol>
			<li>Filter purchased FCSB (see Table of Materials) through a 0.22 &#956;m filter.</li>
			<li>Store the remaining solution at 4 &#176;C.</li>
		</ol>
		</li>
		<li>5% Blocking Buffer (BB) 
		NOTE: Prepare fresh each time.
		<ol>
			<li>Add 0.5 g of BSA to 10 mL of sterile FCSB.</li>
			<li>Vortex to thoroughly mix the sample and leave on ice until use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Antibody conjugation&#8203;
	<ol>
		<li>Conjugate human norovirus GII antibody (see Table of Materials) with r-phycoerythrin (PE) using an R-PE antibody labeling kit per the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>Store the conjugated antibody in the dark at 4 &#176;C for future use in VLP-bacterium attachment assay analysis. 
		NOTE: The primary antibody should be titrated before use in the VLP-bacteria attachment assay to determine the proper concentration necessary for flow cytometry analysis. Antibody titration should be performed every time the conjugation reaction is performed and for each bacterium used in the attachment assay.</li>
	</ol>
	</li>
	<li>Antibody titration
	<ol>
		<li>Dilute the conjugated anti-human norovirus GII antibody 100-fold, with a starting dilution of 1:100 and an ending dilution of 1:600 (six dilutions in total).</li>
		<li>Perform a virus attachment assay as described below with the following exception: in step 1.5.7, resuspend the bacterial pellet in 350 &#181;L of 5% BB.</li>
		<li>Divide the sample into seven 50 &#181;L aliquots.</li>
		<li>Add 50 &#181;L of each antibody dilution to one tube.</li>
		<li>Add 50 &#181;L of 5% BB to the seventh tube as an unstained control.</li>
		<li>Perform flow cytometry on all samples as described below.</li>
		<li>Compare diluted antibody samples to unstained controls and to each other. The lowest antibody concentration that does not result in a decrease or loss of positive signal should be chosen for subsequent assays.</li>
	</ol>
	</li>
	<li>Preparation of bacteria
	<ol>
		<li>Grow the bacteria in 40 mL of appropriate liquid medium under proper atmospheric conditions until the bacteria are in the stationary phase.
		<ol>
			<li>Grow Enterobacter cloacae cultures aerobically overnight in Luria Broth at 37 &#176;C, shaking at 200 rpm to reach the stationary phase.</li>
			<li>Grow Lactobacillus gasseri cultures in De Man, Rogosa, and Sharpe (MRS) broth in black screw cap tubes without shaking in a water bath set to 37 &#176;C for 18 h to reach the stationary phase.</li>
		</ol>
		</li>
		<li>Transfer stationary phase cultures to clean 50 mL conical tubes and centrifuge samples at 2,288 x g for 10 min to pellet the bacteria.</li>
		<li>Remove the supernatant and resuspend it in 13 mL of sterile 1x PBS. Repeat this wash step for a total of two washes.</li>
		<li>Centrifuge the samples again, remove the supernatant, and resuspend the samples in 20 mL of 1x PBS. 
		NOTE: If the cell pellet is small, the resuspension volume can be decreased.</li>
		<li>Measure the OD600 of the culture.</li>
		<li>Using a standard curve, determine the colony-forming unit (CFU) per mL of the washed culture.</li>
		<li>Dilute the bacteria with 1x PBS to adjust the culture concentration to 1 x 108 CFU/mL.</li>
		<li>Transfer 1 mL of the 1 x 108 CFU/mL bacterial culture to the required 1.5 mL centrifuge tubes.</li>
		<li>Centrifuge the tubes at 10,000 x g for 5 min.</li>
		<li>Resuspend the bacterial pellet in 1 mL of sterile 5% BSA.</li>
		<li>Incubate the tubes for 1 h at 37 &#176;C with constant rotation.</li>
	</ol>
	</li>
	<li>Virus attachment&#8203;
	<ol>
		<li>Working inside a BSL-2 biosafety cabinet, add 10 &#181;g of human norovirus (HuNoV) VLPs (see Table of Materials) to each tube containing bacteria resuspended in PBS and mix thoroughly by pipetting. 
		NOTE: VLP concentration differs with each VLP preparation. Therefore, the volume added to bacteria will vary between preparation batches and should be adjusted accordingly so that 10 &#181;g is added to each tube in the experiment. For bacteria-only controls (e.g., samples without VLP), add a volume of PBS that equals the volume of VLP added to experimental samples.</li>
		<li>Incubate tubes for 1 h at 37 &#176;C with constant rotation. 
		&#8203;NOTE: A tube revolver (see Table of Materials) set to 40 rpm is used during incubation.</li>
		<li>After incubation, centrifuge the samples at 10,000 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the bacterial pellet in 1 mL of PBS.</li>
		<li>Repeat the wash steps 1.5.3 and 1.5.4.</li>
		<li>Centrifuge the samples at 10,000 x g for 5 min.</li>
		<li>Discard supernatant and resuspend bacterial pellet in 150 &#181;L of 5% BB.</li>
	</ol>
	</li>
	<li>Antibody staining
	<ol>
		<li>Prepare fresh 5% BB. 
		&#8203;NOTE: All work using the fluorescently tagged antibody should be performed in the dark, and the antibody, BB, and FCSB should be kept on ice.</li>
		<li>Dilute human norovirus GII antibody 1:125 for E. cloacae samples and 1:150 for L. gasseri samples in 5% BB, preparing 50 &#181;L of diluted antibody per sample.</li>
		<li>Dilute the isotype antibody in the same way the human norovirus GII antibody was diluted for each bacterium in 5% BB, preparing 50 &#181;L of diluted isotype control per sample.</li>
		<li>Divide each attachment assay sample from step 1.3.7 into three 50 &#181;L aliquots by transferring them into clean 1.5 mL centrifuge tubes.</li>
		<li>To the first set of samples, add 50 &#181;L of BB. This set will be the unstained (Uns) controls.</li>
		<li>Add 50 &#181;L of the GII antibody dilution to the second set. This results in a final antibody concentration of 1:250 for E. cloacae and 1:300 for L. gasseri. This sample set will be the stained (AB) samples.</li>
		<li>Add 50 &#181;L of the diluted isotype antibody to the third set. This set will be the isotype controls (IC). Mix well by pipetting. Incubate the samples on ice and in the dark for 30 min.</li>
		<li>Centrifuge all samples at 10,000 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the samples in 100 &#181;L of FCSB.</li>
		<li>Centrifuge all samples at 10,000 x g for 5 min. Discard the supernatant and resuspend the samples in 100 &#181;L of FCSB.</li>
		<li>Centrifuge all samples at 10,000 x g for 5 min.</li>
		<li>Discard the supernatant and resuspend the samples in 150 &#181;L of FCSB.</li>
		<li>Transfer each sample to an FCSB tube containing 400 &#181;L of FCSB buffer for a total volume of 550 &#181;L.</li>
		<li>Keep samples at 4 &#176;C until they are analyzed by flow cytometry. 
		NOTE: Flow cytometry is performed within 4 h of antibody staining.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5ml Polystrene Round-Bottom Tubes with Cell-Strainer Cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>After antibody staining, sample are transferred into tubes for flow cytometry analysis.</td>
		</tr>
		<tr>
			<td>AnaeroPack</td>
			<td>Thermo Scientific</td>
			<td>R681001</td>
			<td>Anaerobic gas pack used for culture of Lactobacillus gasseri</td>
		</tr>
		<tr>
			<td>BD FacsDiva software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR Fortessa flow cytometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Bioreagents</td>
			<td>BP1605</td>
			<td>Used for flow cytometry</td>
		</tr>
		<tr>
			<td>Flow Cytometry Stain Buffer (FCSB)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
			<td>Used for flow cytometry</td>
		</tr>
		<tr>
			<td>Mouse IgG2b kappa Isotype Control (eBMG2b), PE, eBioscience</td>
			<td>Thermo Fisher Scientific</td>
			<td>12473281</td>
			<td>Isotype control. This antibody is purchased in the conjugated form from the manufacturer.</td>
		</tr>
		<tr>
			<td>MRS Powder</td>
			<td>BD Biosciences</td>
			<td>288130</td>
			<td>Used for media preparation and to culture Lactobacillus gasseri.</td>
		</tr>
		<tr>
			<td>Norovirus capsid G2 Monoclonal Antibody (L34D)</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-18241</td>
			<td>Norovirus GII antibody. This antibody is only available in the unconjugated form and thus must be fluorescently conjugated prior to use in the outlined flow cytometry assays. In this protocol, PE was the chosen fluor, however, other fluorescent molecules can be chosen as best suits the flow cytometer being used by the researcher.</td>
		</tr>
		<tr>
			<td>Norovirus GII.4 VLP</td>
			<td>Creative Biostructure</td>
			<td>CBS-V700</td>
			<td>Human norovirus virus like particle, VLPs were generated using the baculovirus system and resuspended in phosphate buffered saline with 10% glycerol. The authors performed independent nanosight tracking analysis to determine the particle concentration of the VLPs. The concentration is approximately 1011 VLPs per milliliter. Based on the protein concentration of the VLPs, approximately 200 particles are added per bacterium in VLP attachment assays.</td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Fisher Bioreagents</td>
			<td>BP665</td>
			<td>Dilute to 1X prior to use.</td>
		</tr>
		<tr>
			<td>SiteClick R-PE Antibody Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>S10467</td>
			<td>Conjugation kit used for labling of unconjugated antibody.</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Tube Revolver</td>
			<td>ThermoFisher Scientific</td>
			<td>88881001</td>
			<td>Used in virus:bacterium attachment assay. Set to max speed (40 rpm).</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td>Used for media preparation</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A7002</td>
			<td>Used for media preparation</td>
		</tr>
	</tbody>
</table>

an assay to quantify the binding of human norovirus vlps to intestinal bacteria

Source: Madrigal, J. L., et al. <a href="https://app.jove.com/v/61048">Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry.</a> J. Vis. Exp. (2020).
This video demonstrates an assay for quantifying human norovirus virus-like particles (VLPs) binding to human intestinal bacteria. The bacterial suspensions are mixed with the VLPs and incubated to allow the formation of VLP-bacteria complexes. The complexes are then labeled with fluorophore-tagged VLP-specific antibodies, followed by flow cytometry-based visualization and quantification to identify the percentage of VLP-bound bacteria.

An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

1. VLP-bacteria Attachment Assay
CAUTION: Human norovirus VLPs are a biosafety level (BSL)-2 hazard, and all work involving VLPs should be performed in a ...

Take a tube containing a suspension of human intestinal bacteria.
Add human norovirus virus-like particles, or VLPs. The VLPs contain an intact viral capsid but lack genomic RNA, rendering them non-infectious to humans.
During incubation, the VLP capsid proteins bind to specific carbohydrates expressed on the bacterial surface, forming VLP-bacteria complexes.
Centrifuge to pellet the complexes and discard the unbound VLPs.
Introduce a protein-containing blocking solution to block non-specific antibody binding sites, reducing background staining in subsequent steps.
Add fluorescently labeled VLP-specific antibodies and incubate.
The antibodies bind to their target sites on the VLP capsid, labeling the VLP-bacteria complexes.
Centrifuge to pellet the complexes. Discard unbound antibodies and resuspend the pellet in a flow cytometry buffer.
Using a flow cytometer, detect fluorescence signals emitted by VLP-bacteria complexes, allowing for the quantification of the percentage of bound bacteria in the sample.

an-assay-to-quantify-binding-human-norovirus-vlps-to-intestinal

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80 vistas. Fuente: Madrigal, J. L., et al. Cuantificación de partículas similares al virus del norovirus humano que se unen a bacterias comensales mediante citometría de flujo. J. Vis. Exp. (2020). Este video muestra un ensayo para cuantificar las partículas similares al virus del norovirus humano (VLP) que se unen a las bacterias intestinales humanas. Las suspensiones bacterianas se mezclan con las VLP y se incuban para permitir la formación de complejos VLP-bacterias. A continuación, los complejos...

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Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

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Establecimiento de la infección Viral y análisis de la interacción huésped-Virus en Drosophila Melanogaster

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This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

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Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Detección automatizada y de alto rendimiento de la adherencia bacteriana a las células huésped

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An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

Transcripción

An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria