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 g of yeast extract and 10 g of sodium chloride (NaCl) in 1 L of de-ionized (DI) water (see Table of Materials). Mix all media thoroughly and sterilize by autoclaving for 30 min.</li>
		<li>Prepare solid medium by dissolving 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar in 1 L of DI water. Mix all media thoroughly and sterilize by autoclaving for 30 min.</li>
	</ol>
	</li>
	<li>Lactobacillus gasseri growth media
	<ol>
		<li>Prepare liquid medium by dissolving 55 g of De Man, Rogosa and Sharpe (MRS; see Table of Materials) powder in 1 L of DI water. Use medium within one month of preparation. To sterilize, heat medium for 15 min until boiling followed by autoclaving for 15 min.</li>
		<li>Prepare solid medium by dissolving 55 g of MRS powder and 15 g of agar in 1 L of DI water. To sterilize, heat medium for 15 min until boiling followed by autoclaving for 15 min.</li>
	</ol>
	</li>
</ol>
2. Establishing a Standard Curve Correlating Optical Density (OD) and Bacteria Concentration
<ol>
	<li>Inoculate 5 mL of appropriate liquid medium with a single, isolated colony from an agar plate (E. cloacae) or directly from frozen glycerol stock (L. gasseri).</li>
	<li>Grow the bacterium overnight, under proper atmospheric conditions (Enterobacter cloacae: 37 &#176;C with aerobic shaking at 200 rpm; L. gasseri: 37 &#176;C water bath with no shaking in an airtight container).</li>
	<li>Transfer 2 x 1.3 mL aliquots of overnight culture into two separate 1.5 mL centrifuge tubes and centrifuge bacteria at 10,000 x g for 5 min.</li>
	<li>Remove the supernatant and wash samples with 1 mL of sterile 1x phosphate buffered saline (PBS). Repeat this wash step for a total of 2 washes.</li>
	<li>Centrifuge the samples again at 10,000 x g for 5 min, remove the supernatant and resuspend in 1.3 mL of sterile 1x PBS.</li>
	<li>Beginning with 0.5 mL of washed culture, serially dilute the bacteria in 1x PBS from 10-1 to 10-4.</li>
	<li>Using a spectrophotometer, measure the optical density of the washed, undiluted culture and each of the four dilutions at 600 nm (OD600).</li>
	<li>Perform 10-fold serial dilutions in 1x PBS for each dilution from step 2.6. Spread plate 100 &#181;L of the last 3 dilutions for each series onto appropriate solid medium to determine the number of colony forming units per milliliter (CFU/mL) of each sample. Plate each dilution in triplicate.</li>
	<li>Allow the plates to dry at room temperature for 5 min. Invert the plates and incubate under the appropriate atmospheric conditions (E. cloacae: aerobic at 37 &#176;C, incubate plates overnight; L. gasseri: anaerobic at 37 &#176;C, incubate plates for 48 h in an airtight container with anerobic air sachets (see Table of Materials)). 
	NOTE: Use 1 anaerobic air sachet per 2.5 L jar or 3 sachets per 7.0 L jar (see Table of Materials).</li>
	<li>Use plate counts to determine CFU/mL for each of the 5 samples (i.e., undiluted, 10-1, 10-2, 10-3, 10-4). Generate a standard curve comparing OD600 vs CFU/mL. The equation for this line will be used in VLP-bacteria attachment assays to determine CFU/mL based on OD600.</li>
</ol>
3. 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, prior to the attachment assay, should be performed using safety conditions appropriate for the organism.
<ol>
	<li>Reagent preparation
	<ol>
		<li>1x phosphate buffered saline (PBS)
		<ol>
			<li>Prepare 1 L of 10x PBS by dissolving an entire packet in 1 L of 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 store 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 solution by vortexing or on a stir plate using a metal stir bar 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 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 fully mix sample and leave on ice until use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Antibody conjugation
	<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 prior to 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 3.5.7 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 use in subsequent assays.</li>
	</ol>
	</li>
	<li>Preparation of bacteria
	<ol>
		<li>Grow the bacteria in 40 mL of appropriate liquid medium under appropriate atmospheric conditions until the bacteria are in stationary phase.
		<ol>
			<li>Grow E. cloacae cultures aerobically overnight in Luria Broth at 37 &#176;C, shaking at 200 rpm to reach stationary phase.</li>
			<li>Grow L. gasseri cultures in MRS broth in black screw cap tubes without shaking in a water bath set to 37 &#176;C for 18 h to reach 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 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 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 the previously prepared standard curve, determine the CFU per mL of the washed culture.</li>
		<li>Dilute the bacteria as needed with 1x PBS to adjust 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 number of 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
	<ol>
		<li>Working inside a BSL-2 biosafety cabinet, add 10 &#181;g of 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. 
		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 supernatant and resuspend bacterial pellet in 1 mL of PBS.</li>
		<li>Repeat the wash steps 3.5.3 and 3.5.4.</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. 
		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 3.3.7 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>To the second set, add 50 &#181;L of the GII antibody dilution. 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>To the third set, add 50 &#181;L of the diluted isotype antibody. 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 a 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>
4. Flow Cytometry
NOTE: The voltage settings described below are based on the flow cytometer and software listed in the Table of Materials and will likely vary with different flow cytometers. Settings should be optimized for each bacterium. Ensure that the axes for all graphs are in biexponential phase.
<ol>
	<li>Setting up the workspace 
	NOTE: The set-up for plots used to establish the workspace differ from those used in gating and data analysis. The purpose of setting up the workspace is to visualize the cell population, ensure there is no excessive clumping of the bacterial cells that might impact downstream analysis and to distinguish between stained and unstained cells while data is being collected.
	<ol>
		<li>In order to ensure bacteria are not clumping together, set up a series of density plots.
		<ol>
			<li>Generate a forward scatter area (FSC-A) versus side scatter area (SSC-A) to visualize the total population.</li>
			<li>Set up plots to evaluate single cells versus cell clumps by creating two graphs that compare both forward (FSC-W) and side scatter width (SSC-W) to forward (FSC-H) and side scatter height (SSC-H), respectively.</li>
			<li>Create a plot that shows only the PE positive population (PE-A vs. SSC-A).</li>
		</ol>
		</li>
		<li>Set baseline voltage to 500 for E. cloacae and 340 for L. gasseri. These will be further adjusted later.</li>
		<li>Set the total events counted to 10,000 events.</li>
	</ol>
	</li>
	<li>Running samples
	<ol>
		<li>Create three separate histogram plots that show count plotted against SSC-A, FSC-A or PE-A.</li>
		<li>Place an unstained sample on the flow cytometer and acquire the sample events, ensuring that the maximum peak on the histogram for SSC-A is within 102 to 103 and the maximum peak on the histogram for FSC-A is between 104 to 105 on the x-axis. If not, peaks do not fall within the specified ranges, adjust the FSC and SSC voltages accordingly.</li>
		<li>Place a stained sample (AB) on the flow cytometer and adjust the voltage PE to make the maximum peak past 103 on the x-axis.</li>
		<li>Based on these measurements, set the positive PE gate and set the gate on the PE-A versus SSC-A density graph.</li>
		<li>Run all samples under the determined settings at low or medium speed.</li>
	</ol>
	</li>
</ol>

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The authors do not have any conflicts of interest.

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 ...

quantifying-human-norovirus-virus-like-particles-binding-to-commensal

Research

JoVE Journal

Immunology and Infection

7.7K Views.  University of Florida. 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.

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

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 ...

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

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 ...