We would like to thank Sutonuka Bhar and Chanel Mosby-Haundrup for their critical review of the written manuscript, as well as Alfonso Carrillo for assistance with generating bacterial standard curves. This work is funded in part by a grant from the National Institute of Health (R21AI140012) and by a seed grant from the University of Florida, Institute of Food and Agricultural Sciences.

NOTE: The bacterial growth conditions outlined in the protocol are standard culture conditions for Enterobacter cloacae and Lactobacillus gasseri. To perform the virus:bacteria attachment assay with other bacterial species, the chosen bacteria should be cultured under standard conditions appropriate for the bacterium.
1. Preparing Bacterial Growth Medium
<ol>
	<li>Enterobacter cloacae growth media
	<ol>
		<li>Prepare liquid medium by dissolving 10 g of tryptone, 5 ...

<ol>
	<li>Hall, A. J., Glass, R. I., Parashar, U. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+global+burden+of+noroviruses+and+opportunities+for+prevention.">New insights into the global burden of noroviruses and opportunities for prevention.</a> Expert Review of Vaccines. 15 (8), 949-951 (2016).</li><li>Jones, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+bacteria+promote+human+and+mouse+norovirus+infection+of+B+cells.">Enteric bacteria promote human and mouse norovirus infection of B cells.</a> Science. 346 (6210), 755-759 (2014).</li><li>Baldridge, M. T., 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=Commensal+microbes+and+interferon-lambda+determine+persistence+of+enteric+murine+norovirus+infection.">Commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection.</a> Science. 347 (6219), 266-269 (2015).</li><li>Lei, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterobacter+cloacae+inhibits+human+norovirus+infectivity+in+gnotobiotic+pigs.">Enterobacter cloacae inhibits human norovirus infectivity in gnotobiotic pigs.</a> Scientific reports. 6, 25017 (2016).</li><li>Lei, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Protective+Efficacy+of+Probiotics+and+Rice+Bran+against+Human+Norovirus+Infection+and+Diarrhea+in+Gnotobiotic+Pigs.">High Protective Efficacy of Probiotics and Rice Bran against Human Norovirus Infection and Diarrhea in Gnotobiotic Pigs.</a> Frontiers in Microbiology. 7, 1699 (2016).</li><li>Rodr&#237;guez-D&#237;az, J., 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=Relevance+of+secretor+status+genotype+and+microbiota+composition+in+susceptibility+to+rotavirus+and+norovirus+infections+in+humans.">Relevance of secretor status genotype and microbiota composition in susceptibility to rotavirus and norovirus infections in humans.</a> Scientific Reports. 7, 45559 (2017).</li><li>Erickson, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+Facilitate+Enteric+Virus+Co-infection+of+Mammalian+Cells+and+Promote+Genetic+Recombination.">Bacteria Facilitate Enteric Virus Co-infection of Mammalian Cells and Promote Genetic Recombination.</a> Cell Host Microbe. 23 (1), 77-88 (2018).</li><li>Kuss, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+microbiota+promote+enteric+virus+replication+and+systemic+pathogenesis.">Intestinal microbiota promote enteric virus replication and systemic pathogenesis.</a> Science. 334 (6053), 249-252 (2011).</li><li>Robinson, C. M., Jesudhasan, P. R., Pfeiffer, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharide+binding+enhances+virion+stability+and+promotes+environmental+fitness+of+an+enteric+virus.">Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus.</a> Cell Host Microbe. 15 (1), 36-46 (2014).</li><li>Berger, A. K., Yi, H., Kearns, D. B., Mainou, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+and+bacterial+envelope+components+enhance+mammalian+reovirus+thermostability.">Bacteria and bacterial envelope components enhance mammalian reovirus thermostability.</a> PLOS Pathogens. 13 (12), 1006768 (2017).</li><li>Miura, T., 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=Histo-blood+group+antigen-like+substances+of+human+enteric+bacteria+as+specific+adsorbents+for+human+noroviruses.">Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.</a> Journal of Virology. 87 (17), 9441-9451 (2013).</li><li>Almand, E. A., Moore, M. D., Outlaw, J., Jaykus, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+norovirus+binding+to+select+bacteria+representative+of+the+human+gut+microbiota.">Human norovirus binding to select bacteria representative of the human gut microbiota.</a> PLOS One. 12 (3), (2017).</li><li>Robinson, C. M., Woods Acevedo, M. A., McCune, B. T., Pfeiffer, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Related+enteric+viruses+have+different+requirements+for+host+microbiota+in+mice.">Related enteric viruses have different requirements for host microbiota in mice.</a> Journal of Virology. , (2019).</li><li>Ettayebi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+human+noroviruses+in+stem+cell-derived+human+enteroids.">Replication of human noroviruses in stem cell-derived human enteroids.</a> Science. 353 (6306), 1387-1393 (2016).</li><li>Van Dycke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+human+norovirus+replication+model+in+zebrafish+larvae.">A robust human norovirus replication model in zebrafish larvae.</a> PLOS Pathogens. 15 (9), 1008009 (2019).</li><li>Li, D., Breiman, A., le Pendu, J., Uyttendaele, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+to+histo-blood+group+antigen-expressing+bacteria+protects+human+norovirus+from+acute+heat+stress.">Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress.</a> Frontiers in Microbiology. 6, 659 (2015).</li><li>Almand, E. A., Moore, M. D., Jaykus, L. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+norovirus+binding+to+gut-associated+bacterial+ligands.">Characterization of human norovirus binding to gut-associated bacterial ligands.</a> BMC Research Notes. 12 (1), 607 (2019).</li><li>Debbink, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-host+evolution+results+in+antigenically+distinct+GII.4+noroviruses.">Within-host evolution results in antigenically distinct GII.4 noroviruses.</a> Journal of Virology. 88 (13), 7244-7255 (2014).</li><li>Harrington, P. R., Lindesmith, L., Yount, B., Moe, C. L., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+Norwalk+virus-like+particles+to+ABH+histo-blood+group+antigens+is+blocked+by+antisera+from+infected+human+volunteers+or+experimentally+vaccinated+mice.">Binding of Norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice.</a> Journal of Virology. 76 (23), 12335-12343 (2002).</li><li>Harrington, P. R., Vinje, J., Moe, C. L., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+capture+with+histo-blood+group+antigens+reveals+novel+virus-ligand+interactions.">Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions.</a> Journal of Virology. 78 (6), 3035-3045 (2004).</li><li>Mallory, M. L., Lindesmith, L. C., Graham, R. L., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GII.4+Human+Norovirus:+Surveying+the+Antigenic+Landscape.">GII.4 Human Norovirus: Surveying the Antigenic Landscape.</a> Viruses. 11 (2), (2019).</li><li>Prasad, B. V., 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=X-ray+crystallographic+structure+of+the+Norwalk+virus+capsid.">X-ray crystallographic structure of the Norwalk virus capsid.</a> Science. 286 (5438), 287-290 (1999).</li><li>Baric, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+self-assembly+of+norwalk+virus+capsid+protein+from+venezuelan+equine+encephalitis+virus+replicons.">Expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons.</a> Journal of Virology. 76 (6), 3023-3030 (2002).</li><li>Rubio-del-Campo, A., 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=Noroviral+p-particles+as+an+in+vitro+model+to+assess+the+interactions+of+noroviruses+with+probiotics.">Noroviral p-particles as an in vitro model to assess the interactions of noroviruses with probiotics.</a> PLOS One. 9 (2), 89586 (2014).</li><li>Tan, M., 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=Terminal+modifications+of+norovirus+P+domain+resulted+in+a+new+type+of+subviral+particles,+the+small+P+particles.">Terminal modifications of norovirus P domain resulted in a new type of subviral particles, the small P particles.</a> Virology. 410 (2), 345-352 (2011).</li></ol>

The ability to quantify binding of enteric viruses to bacteria is a critical first step for elucidating the mechanisms by which these bacteria alter viral infection. The methods described herein have been optimized to measure human norovirus VLP interactions with both E. cloacae (gram-negative bacterium) and L. gasseri (a gram-positive bacterium), but can be adapted for use with any mammalian virus and bacterium of interest. While VLPs are an ideal alternative to live vi...

Human noroviruses (HuNoVs) are the leading cause of gastrointestinal illness worldwide, responsible for 685 million infections and over 200,000 deaths each year1. As with other enteric viruses, the presence of commensal bacteria has been shown to enhance infection of this pathogen as well as its surrogate virus, murine norovirus2,3. There are also conflicting reports that bacteria may inhibit infection by human norovirus4,5,6. For several viruses, direct interaction between the virus and bacteria appear to underlie the mechanisms that impact viral infection2,7,8,9,10, and it has been shown through electron microscopy that human noroviruses bind directly to the surfaces of bacteria11,12. Therefore, characterizing these interactions has become critical to determining the mechanisms by which bacteria impact viral infection. This characterization has classically begun with quantifying viral binding to an array of bacterial species that are components of the host microbiome7,12,13. These attachment assays not only reveal the amount of virus bound to bacteria, but also aid in determining the impact of this interaction on viral fitness and survival.
To quantify viral attachment, traditionally employed methods include PCR-based assays which quantify viral genomes12 or the generation of radiolabeled virus and the use of scintillation counting to quantify viral particles7,8,9,13. The use of these methods generally require access to high-titer virus stocks and in vitro cultivation techniques with which to generate them. While several culture systems for human norovirus now exist2,14,15, none support the robust replication required to generate these highly concentrated stocks which restricts or eliminates the use of PCR and scintillation counting to quantify human norovirus/bacterial interactions.
To circumvent this issue, virus-like particles (VLPs) can be used as a surrogate to live virus to investigate interactions between human norovirus and bacteria16,17. VLPs are non-infectious particles that closely resemble the virus from which they are derived. In the case of human norovirus, these particles are generated from the expression of the VP1 (and sometime the VP2) protein, which self-assemble to create intact viral capsids lacking genetic material (i.e., RNA for noroviruses). These VLPs have been well characterized, are structurally and antigenically similar to the wild-type viruses from which they are derived18,19,20,21,22,23. Therefore, VLPs serve as an ideal surrogate for investigating the surface interactions between human norovirus and commensal bacteria. Given that VLPs lack genetic material, PCR-based assays cannot be used to quantify viral binding. An antibody-based flow cytometry method was previously described and able to detect low levels of VLP binding to bacteria in a semi-quantitative manner16. This method was optimized to allow for accurate quantification of human norovirus VLP binding to both gram-negative and gram-positive commensal bacteria16.

<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>Agar</td>
			<td>Sigma</td>
			<td>A7002</td>
			<td>Used for media preparation</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>
	</tbody>
</table>

quantifying human norovirus virus-like particles binding to commensal bacteria using flow cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The gating strategies used to quantify human norovirus VLP binding to commensal bacteria are shown in Figure 1. Representative density dot provides an overview of how samples were gated to eliminate cellular debris and cell clumps so VLP attachment was determined on singlet populations (Figure 1A). Representative histograms demonstrate low levels of anti-norovirus antibody signal in bacteria only samples lacking ...

Watch this Scientific Journal Video about Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry at JoVE.com

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Virus-bacterial interactions can be important for successful viral infection and stimulation of a host immune response. But currently, highly concentrated and purified stock of human norovirus cannot be produced in the lab. This technique can be used with any virus that binds to bacteria, for which either virus-like particles or live virus are available.It enables us to quantify interactions between these viruses and bacteria. Inoculate five milliliters of liquid medium with a single isolated colony of Enterobacter cloacae from an agar plate directly from frozen glycerol stock. Grow the bacteria overnight.The following day, transfer 1.3 milliliter aliquots of the culture into two separate 1.5 milliliter centrifuge tubes. Centrifuge the tubes at 10, 000 x g for five minutes. Remove the supernatants, then wash the samples twice using one milliliter of sterile 1X PBS for each wash.Centrifuge the samples again at 10, 000 x g for five minutes. Remove the supernatant and resuspend the pellet in 1.3 milliliters of sterile 1X PBS. Beginning with 0.5 milliliters of washed culture, serially dilute the bacteria in 1X PBS from 10 to the minus one to 10 to the minus four.Using a spectrophotometer, measure the optical density of the washed undiluted culture and each of the four dilutions at 600 nanometers. Perform 10-fold serial dilutions in 1X PBS for each of the previous dilutions. Spread 100 microliters of the last three dilutions for each series onto agar plates to determine the number of colony forming units per milliliter for each sample.Allow the plates to dry at room temperature for five minutes. Invert the plates and incubate them in the incubator. After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL-2 biosafety cabinet.Virus-like particles for human norovirus are listed as BSL-2 pathogens and all work perform using VLP should be conducted in a certified biosafety cabinet. Add 10 micrograms of human norovirus VLPs to each tube of bacteria and mix thoroughly by pipetting. Incubate the tubes for one hour at 37 degree Celsius with constant rotation.After incubation, centrifuge the tubes at 10, 000 x g for five minutes. Aspirate and discard the supernatant and resuspend the bacterial pellet in one milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10, 000 X g for five minutes.Discard the supernatant and resuspend the VLP-bacteria pellet in 150 microliters of 5%blocking buffer. A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment and add one treatment all at once to the tubes.For each bacterial sample, prepare 50 microliters of diluted human norovirus GII antibody. Using 5%blocking buffer, dilute the antibody one to 125 for E.cloacae samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios.Divide each attachment assay sample into 350 microliter aliquots by transferring them into clean 1.5 milliliter centrifuge tubes. To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the GII antibody dilution to the second set of aliquots.Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots. Incubate all the samples on ice and in the dark for 30 minutes. Then centrifuge all the samples at 10, 000 X g for five minutes.Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10, 000 X g for five minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.After centrifuging the samples one final time and discarding the supernatants, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degree Celsius until they are analyzed by flow cytometry.VLP attachment was measured using flow cytometry. First, density plots were used to gate out cellular debris, followed by subsequent dot plot gating to remove bacterial doublets and cellular clumps. Representative histograms demonstrate a lack of PE signal in bacterial only samples and a shift in PE signal intensity in VLP:bacterium samples compared to unstained and isotype controls.After one hour of incubation with 10 micrograms of VLP, flow cytometry detected particle binding to both E.colacae and L.gasseri. To determine the limit of binding quantification for this assay, a dilution series of the VLPs was generated prior to addition to the bacteria. Reductions in the amount of VLP resulted in corresponding reductions to the percent bacteria bound by VLP.Knowing the virus:bacterium ratio and keeping the ratio consistent between experiments is important in interpreting results. Bacterial and viral mutants can be generated to specifically determine the microbial surface structures that mediate this interaction. This technique allows researchers to answer questions regarding circumstances and conditions that impact norovirus-bacterial interactions, including how changes in virus genotype, bacterial growth conditions, and changes in bacterial surface structures alter viral binding.

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Immunology and Infection

7.7K ビュー.  University of Florida. ウイルスと細菌の相互作用は、成功したウイルス感染および宿主免疫応答の刺激のために重要であり得る。しかし、現在、ヒトノロウイルスの高度に濃縮され精製されたストックは、実験室で生産することはできません。この技術は、ウイルス様の粒子または生きたウイルスのいずれかが利用可能な細菌に結合する任意のウイルスで使用することができます。これ?...

ビデオ: Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the...

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

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...

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

Virus-like particles for human norovirus are listed as BSL II pathogens, and all work performed using VLPs should be conducted in a certified biosafety...

detection of vitronectin binding to bacterial surfaces using flow cytometry

This video demonstrates the detection of vitronectin&#39;s interaction with bacterial surface proteins. The flow-cytometry analysis measures the fluorescence signal of fluorescently labeled antibodies bound to vitronectin, confirming its presence at the bacterial surface. This technique provides insights into host-pathogen interactions and potential bacterial infection...

Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry

expression and purification of virus-like particles for vaccination

Expression and Purification of Virus-like Particles for Vaccination

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible...

swab-based conjunctival commensal bacteria isolation: a technique to isolate commensal bacteria from conjunctiva of murine model

This video describes a swab-based technique to isolate viable commensal bacteria from the ocular conjunctiva. The technique can be used to profile the commensal bacterial microbiome of the conjunctiva, which plays a defensive role against...

Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model

Following wash, add 50 microliters of primary sheep anti-human vitronectin polyclonal antibodies to the bacterial pellet, at a 1 to 100 dilution in PBS/BSA. Incubate the suspension for 1 hour at room temperature. Then, wash the bacteria three times with PBS to remove unbound...

an assay for quantifying protein-rna binding in bacteria

An Assay for Quantifying Protein-RNA Binding in Bacteria

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter...

Uncap a labeled BHI microcentrifuge tube with the dedicated media handling hand and place the tube back in the rack. Dip the cotton-coated tip of the eye swab in the BHI. Then, withdraw the swab from the tube while swirling the tip twice against the inner tube to remove excess liquid and remove...

measuring microglial uptake of intravitreally-delivered fluorescent particles using flow cytometry

This video demonstrates flow cytometry for measuring microglial uptake of fluorescent bioparticles after intravitreal delivery in a mouse eye. Flow cytometry detects live cells expressing CD11b and distinguishes phagocytic microglia cells exhibiting green fluorescence from other myeloid cells, including Ly6C-expressing monocytes and Ly6G-expressing...

Measuring Microglial Uptake of Intravitreally-Delivered Fluorescent Particles Using Flow Cytometry

Transfer the eyeball into PBS and use a second pair of fine 45-degree angled forceps to tear the cornea and sclera apart. The lens and retina will come out intact. Ensure the lens and retina are separated and transfer the retina to a 5.4-milliliter polystyrene test tube containing 2 milliliters of PBS with calcium and...

generation of chikungunya virus-like particles using baculovirus expression system in insect cells

Use trypan blue exclusion to determine if the cell viability has decreased to 70% to 80%. Upon confirmation, transfer the cultures directly from suspension into 50-milliliter conical tubes, and spin the cells down. Collect the supernatants, and filter them through a 0.22-micron pore membrane before...

Generation of Chikungunya Virus-Like Particles Using Baculovirus Expression System in Insect Cells

This video shows how virus-like particles, VLPs are generated by infecting insect cells with a recombinant baculovirus carrying the chikungunya virus gene cassette. The recombinant virus infects cells, leading to VLP formation, assembly, and release into the culture medium for potential use in vaccine...

a simple flow cytometry based assay to determine in vitro antibody dependent enhancement of dengue virus using zika virus convalescent serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

making conjugation-induced fluorescent pegylated virus-like particles by dibromomaleimide-disulfide chemistry

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside...

measuring phagocytosis of aspergillus fumigatus conidia by human leukocytes using flow cytometry

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

modeling the lifecycle of ebola virus under biosafety level 2 conditions with virus-like particles containing tetracistronic minigenomes

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus...

the virus-like particles capture assay: a method to isolate antigen-displaying vlps from a sample using neutralizing antibody-conjugated magnetic beads

In this video, we demonstrate an immunoprecipitation assay, called the VLP capture assay, to isolate antigen-displaying virus-like particles, or VLPs, from a sample. The antigen binds with specific antibodies bound to magnetic beads, forming an immobilized antigen-antibody complex that is later separated and stored for further...

The Virus-Like Particles Capture Assay: A Method to Isolate Antigen-Displaying VLPs from a Sample Using Neutralizing Antibody-Conjugated Magnetic Beads

quantifying immune cell infiltration in uropathogenic e. coli-infected mouse bladder tissue using flow cytometry

In this video, we demonstrate a flow-cytometry-based technique to quantify CD45+ immune cells in Escherichia coli-infected mouse bladder to measure immune cell...

Quantifying Immune Cell Infiltration in Uropathogenic E. coli-Infected Mouse Bladder Tissue using Flow Cytometry

a technique for the generation of influenza virus-like particles via a mammalian system

This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors &#8212; encoding different viral structural proteins &#8212; in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell...

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...