This work was supported by a grant from the Swiss National Science Foundation (31003A_135278) to SSt. MOP is the beneficiary of a doctoral grant from the AXA Research Fund. We thank Patricia Nigg for help with the design of Figure 1.

Note before start: Use laminar flow hood and appropriate biocontainment when working with live virus. Here, we describe growth conditions suitable to culture influenza virus strain A/WSN/33. Multiplicity of infection (MOI) and incubation times may vary depending on virus strain used.
1. Preparation of Biotinylated A/WSN/33 Virus
<ol>
	<li>Seed Madin-Darby canine kindey (MDCK) cells into a T175 cm2 flask using Dulbecco&#8217;s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (Pen/Strep). Culture cells at 37 &#176;C until approximately 70% confluency is reached.</li>
	<li>Before infection, remove medium and wash cells with phosphate-buffered saline (PBS, pH7.0-7.3) by adding a large enough volume to cover the cell monolayer.</li>
	<li>Remove PBS and infect cells with A/WSN/33 with an MOI of 10-4. Dilute virus in PBS supplemented with 0.02 mg Mg2+, 0.01 mg Ca2+, 0.3% bovine serum albumin (BSA), and 1% Pen/Strep (infection-PBS). Use an inoculum of 4 ml for a T175 cm2 flask.</li>
	<li>Incubate cells 1 hr at 37 &#176;C. Remove inoculum by aspiration and pipette 10 ml DMEM supplemented with 0.3% BSA, 20 mM HEPES, and 1% Pen/Strep into the flask.</li>
	<li>Incubate cells at 37 &#176;C for approximately 3 days until cytopathic effect (CPE) is visible, and then harvest supernatant. CPE can be recognized as rounding up of cells followed by detachment and death of cells 14.</li>
	<li>Spin supernatant at 450 x g for 5 min to remove cell debris. Transfer supernatant into a new falcon tube and repeat this step one more time. Discard the pellets.</li>
	<li>Transfer supernatants into ultracentrifuge tubes and underlay supernatants with 5 ml of 30% sucrose diluted in NTE buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1 mM EDTA). Balance tubes before ultracentrifugation and fill up with PBS to reach an end volume of 30 ml. Spin supernatants at 112,398 x g (corresponding to 25,000 rpm for an SW28 rotor) for 90 min in an ultracentrifuge.</li>
	<li>Carefully remove supernatant by pipetting and soak virus pellet overnight at 4 &#176;C in 250 &#181;l PBS. Resuspend pellet by pipetting up and down. Measure protein content of purified virus by Bradford assay 15.</li>
	<li>Use a biotinylation kit to generate biotinylated A/WSN/33. Follow the instruction of the manufacturer.</li>
	<li>In brief, add the appropriate amount of biotin to the purified virus and incubate for 2h on ice. Transfer virus to an ultracentrifuge tube and fill tube up with PBS to reach an end volume of 30 ml. Spin virus at 112,389 xg for 90 min in an ultracentrifuge. Remove supernatant by pipetting and add 250 &#181;l PBS. Soak pellet over night at 4 &#176;C. Aliquot and store the Biotinylated virus stored at -80 &#176;C where it will be stable for at least 24 months. 
	NOTE: It might be helpful to determine the titer of the biotinylated preparation of IAV by standard plaque assay. However, it should be noted that the subsequent protocol will only measure biotinylated virus particles. Remaining unbiotinylated particles due to incomplete biotinylation will contribute to the infectious titer by plaque assay but not to the signal measured in the attachment/internalization assay.</li>
</ol>
2. Determine the Required Amount of Biotinylated Virus &#160;
NOTE: Before the attachment/internalization assay is performed, it is important to determine the amount of biotinylated virus required to infect all cells of a sample.
<ol>
	<li>Culture and trypsinize A549 cells according to the manufacturer&#8217;s instructions. Count the cells using a cell chamber. Pipette 200,000 A549 cells into FACS deepwell tubes. Spin 3 min at 450 x g. Remove supernatant and resuspend pellet with 200 &#181;l PBS. Cool cells down on ice for 10 min.</li>
	<li>Prepare 5-fold serial dilutions of the biotinylated virus stock in infection-PBS.</li>
	<li>Spin tubes containing the cells at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet with 100 &#181;l virus-containing infection-PBS. As mock control use infection-PBS without biotinylated virus. Incubate tubes on ice for 1 hr.</li>
	<li>Spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 &#181;l PBS. Repeat this step one more time.</li>
	<li>For fixation, spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 100 &#181;l of 3.7% paraformaldehyde (PFA). Incubate for 10 min at RT. Then, repeat wash step described in 2.4).</li>
	<li>For permeabilization, spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 100 &#181;l PBS containing 0.5% Triton X-100. Incubate for 6 min at RT. Then, repeat wash step described in 2.4).</li>
	<li>Spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 50 &#181;l 2% BSA diluted in PBS containing STV-Cy3. (Please note, the appropriate concentration of STV-Cy3 should be determined before starting the experiment. Start with a concentration of 1:200 and test 2-fold dilutions up to 1:2,400. In our hands, a dilution of 1:1,000 proved to be optimal.)</li>
	<li>Incubate 1 hr in the dark at RT. Then, repeat washing step described in 2.4 but resuspend the pellet in 200 &#181;l 2% BSA diluted in PBS.</li>
	<li>Analyze samples by flow cytometry 16. Gate on the Cy3-positive population. Choose the lowest concentration of biotinylated virus resulting in the maximum number of Cy3-positive cells in the A549 population.</li>
</ol>
3. Determine the Required Amount of Streptavidin
 
NOTE: The efficiency of blocking the signal derived from extracellular virus determines the detection range of the assay. Therefore, it is important to abrogate the STV-Cy3 signal as much as possible. The required amount of STV needed depends on the amount of biotinylated virus stock used (determined in section 2).
<ol>
	<li>Follow steps 2.1-2.4, but use virus concentration determined in section 2.</li>
	<li>Prepare five-fold serial dilutions of STV diluted in PBS containing 2% BSA and 0.1% sodium azide.</li>
	<li>Spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 50 &#181;l STV dilution. As mock control use PBS containing 2% BSA and 0.1% sodium azide without STV. Incubate tubes for 30 min on ice. Then, repeat wash step described in 2.4.</li>
	<li>Follow steps 2.5-2.9. For the internalization assay, choose the lowest concentration of STV resulting in a strong block of the Cy3-signal (compared to the sample in which no STV was present). For this, the percentage of CY3-positive cells or the mean fluorescence intensity of the Cy3 signal can be used. Ideally, the Cy3 signal of STV-treated samples should be as close to the signal from mock-infected cells as possible.</li>
</ol>
4. Attachment/Internalization Assay
 
NOTE: Make sure to prepare all reagents before starting the assay. For the three control groups presented in the results section (mock infected cells, neuraminidase pre-treated cells and sodium azide treated cells) small alterations to the protocol are required.
<ol>
	<li>Prepare 16 deepwell tubes each containing 200,000 A549 cells. Next to the experimental set consisting of 4 tubes, there are 3 control conditions, each requiring 4 tubes: mock-infected cells, neuraminidase (NA)-pretreated cells and sodium azide-treated cells. Each group consists of 4 tubes labelled as &#8216;0 min&#8217;, &#8216;0 min + STV&#8217;, &#8216;30 min&#8217;, &#8216;30 min + STV&#8217;.</li>
	<li>Spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 &#181;l PBS.</li>
	<li>Spin tubes at 4 &#176;C at 450 x g. Remove supernatant and resuspend pellet in 200 &#181;l DMEM containing 0.3% BSA, 20 mM HEPES and 1% penicillin-streptomycin (incubation medium). Incubate for 30 min at 37 &#176;C. 
	NOTE: For the neuraminidase control: Use bacterial neuraminidase (e.g. sialidase from Vibrio cholera) at a concentration of 200 mU/ml diluted in incubation medium to resuspend the cells.</li>
	<li>Spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 &#181;l PBS. Repeat this step one more time, then cool tubes down on ice for 10 min.</li>
	<li>Spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 100 &#181;l biotinylated virus (use the virus concentration determined in section 2 diluted in infection-PBS). Incubate 1 hr on ice. Then, repeat wash step described in 2.4. 
	NOTE: For the mock-infected control: Use infection-PBS without virus. For the sodium azide control: Use virus diluted in infection-PBS supplemented with 0.1 % sodium azide.</li>
	<li>Processing of the samples after the infection on ice:
	<ol>
		<li>For the &#8216;0 min&#8217; samples, follow step 2.5 and store samples at 4 &#176;C until the other samples are fixed.</li>
		<li>For the &#8216;0 min + STV&#8217; samples, spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 50 &#181;l STV (use the concentration determined in section 3) diluted in PBS containing 2% BSA and 0.1% sodium azide (to prevent internalization while handling the sample). Incubate 30 min on ice. Follow step 2.4-2.5 and store samples at 4 &#176;C.</li>
		<li>For the &#8217;30 min&#8217; samples, spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 &#181;l PBS containing 2% BSA. Incubate 30 min at 37 &#176;C. Follow step 2.4-2.5 and store samples at 4 &#176;C. 
		NOTE: For the sodium azide control: Use PBS supplemented with 2 % BSA and 0.1 % sodium azide for the 30 min incubation at 37 &#176;C.</li>
		<li>For the &#8217;30 min + STV&#8217; samples spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 200 &#181;l PBS containing 2% BSA. Incubate 30 min at 37 &#176;C. Then, spin tubes at 4 &#176;C for 3 min at 450 x g. Remove supernatant and resuspend pellet in 50 &#181;l STV (use the concentration determined in section 3) diluted in PBS containing 2% BSA and 0.1% sodium azide (to prevent internalization while handling the sample). Incubate 30 min on ice. Follow step 2.4-2.5 and store samples at 4 &#176;C. 
		NOTE: For the sodium azide control: Use PBS supplemented with 2 % BSA and 0.1 % sodium azide for the 30 min incubation at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Follow steps 2.6-2.8 and analyze samples by flow cytometry. Determine the percentage of Cy3-positive cells in each sample.</li>
	<li>Calculate the percentage attached virus and the relative amount of internalized virus.
	<ol>
		<li>To calculate the amount of attached virus, use the percentage of Cy3-positive gated cells or the mean fluorescence intensity of the Cy3 signal. For comparison, set untreated cells to 100 % (Figure 4B). 
		NOTE: The percentage of attached virus is described by the &#8220;0 min&#8221; sample.</li>
		<li>To calculate the amount of internalized virus, take the ratio of the &#8220;30 min + STV&#8221; sample to the &#8220;30 min&#8221; sample (Figure 5B). 
		NOTE: As mentioned above, the percentage of Cy3 positive gated cells or the mean fluorescence intensity of the Cy3 signal can be used.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Our protocol describes an easy way of measuring virus attachment and internalization by flow cytometry. It allows the use of labeled wildtype virus which mimics more closely virus infections compared to the use of virus-like particles (VLP). While our protocol has been optimized to measure attachment and internalization of IAV it can easily be adapted for other viruses. In addition, as flow cytometry is used for readout, co-stainings can be easily added to the protocol e.g. to test expression levels following kn...

The entry of influenza A virus (IAV) is a multi-step process that starts with the binding of the virus to receptors on the plasma membrane of target cells 1. The receptor for IAV is sialic acid which is present on a large variety of glycoproteins and glycolipids. The hemagglutinin (HA) protein of IAV which is present in the viral envelope binds to sialic acid and thereby mediates attachment of the viral particles to the plasma membrane of target cells 2. The virus enters the cells via clathrin-mediated endocytosis but also alternative entry pathways, such as macropinocytosis, have been described 3-6. The interaction between HA and sialic acid appears to be sufficient for mediating both, attachment and triggering internalization of viral particles 7. Nevertheless, alternative entry receptors have been proposed and their roles in IAV entry are currently being investigated 1,8,9.
Currently, efforts are made to identify host factors that are involved in the viral life cycle with the aim of developing urgently needed novel antiviral therapies 10-12. Virus entry would be a favorable step for targeting IAV growth in order to block viral infection at its earliest point. Measuring different stages of IAV entry experimentally is challenging as usually large amounts of virus are required to detect incoming virus. In addition, the detection range for changes in virus entry is only linear due to the lack of viral replication. This underscores the need for assays with high sensitivity.
The assay we present here allows the detection of attached virus at the cell surface as well as detection of internalized virus in relation to the total amount of cell-associated virus. The use of biotinylated wildtype virus enables convenient measurement through staining with STV-Cy3 and readout by flow cytometry. Biotinylated virus is cold-bound to cells to allow attachment but prevent internalization of viral particles. Cells can be fixed, permeabilized and stained with STV-Cy3 to measure attached virus. The signal from extracellular, attached virions can be abrogated if a blocking step is applied before fixation in which the cells are incubated with non-labeled streptavidin (STV). In a next step, following attachment of biotinylated IAV, temperature is shifted to 37 &#176;C and internalization of viral particles is allowed to take place. Internalized particles are protected from binding of STV thereby allowing the discrimination between extra- and intracellular viruses.
Per experimental condition, four samples are required: &#8216;0 min&#8217;: The first sample, labeled &#8216;0 min&#8217;, is to measure cold-bound virus at the cell surface. &#8216;0 min + STV&#8217;: The second sample, labeled &#8216;0 min + STV&#8217;, gives the baseline signal intensity of the experiment. Attached virus is blocked with STV and the signal following staining with STV-Cy3 should be much lower compared to the &#8216;0 min&#8217; sample. &#8217;30 min&#8217;: The third sample, &#8217;30 min&#8217; contains attached and internalized virus due to the temperature shift to 37 &#176;C for 30 min. &#8217;30 min + STV&#8217;: The fourth sample, &#8217;30 min + STV&#8217; measures the intracellular fraction of viruses. The STV blocking step is applied after the 30 min incubation period. As a result, viral particles at the cell surface are bound by STV leaving only internalized viruses available for staining with STV-Cy3. The relative amount of internalized virus can be calculated as the ratio of internalized virus (measured in the &#8217;30 min + STV&#8217; sample) divided by the total amount of virus (described by the &#8217;30 min&#8217; sample).
As controls we suggest to include mock-infected cells. The signal following STV-Cy3 staining of mock-infected cells gives the background resulting from the staining protocol. A control for IAV attachment is the pre-treatment of cells with bacterial neuraminidase (NA). NA cleaves off sialic acid from cellular glycoproteins, thereby removing attachment receptors from the cell surface. Sodium azide (SA) is a potent metabolic inhibitor and thereby blocks endocytosis 13. Cells treated with sodium azide should be positive for virus binding but negative for internalization.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966-052</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-163</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>14190-169&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>VWR Calbiochem</td>
			<td>126579</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630-100</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sucrose</td>
			<td>Fluka</td>
			<td>84100</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Biosolve BV</td>
			<td>20092391</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>3680</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-LINK NHS-SS-BIOTIN kit</td>
			<td>Fisher Scientific</td>
			<td><a href="https://extranet.fisher.co.uk/insight2_ch/getProduct.do?productCode=W9971E">W9971E&#160;</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio Rad Protein Bio Assay</td>
			<td>Bio Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>deepwell tubes (1.2 ml microtubes)</td>
			<td>Milian</td>
			<td>82 00 001</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>&#160;Lucerna chem&#160; Electron microscopy sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge tubes</td>
			<td>Hemotec HmbH</td>
			<td>253070</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>STV-Cy3</td>
			<td>Life Technologies</td>
			<td>43-4315</td>
			<td></td>
		</tr>
		<tr>
			<td>STV</td>
			<td>Life Technologies</td>
			<td>43-4302</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium azide</td>
			<td>Fluka</td>
			<td>71290</td>
			<td></td>
		</tr>
		<tr>
			<td>bacterial neuraminidase/sialidase</td>
			<td>Sigma-Aldrich</td>
			<td>N6514-1UN</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring attachment and internalization of influenza a virus in a549 cells by flow cytometry

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 &#176;C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.

A cartoon describing the four different experimental conditions is shown in Figure 1. Results of a representative experiment are presented in Figures 2-5. In the &#8220;0 min&#8221; sample, biotinylated virus is cold-bound to target cells which can be visualized by STV-Cy3 staining (Figures 1 and 2). When a blocking step (in the &#8220;0 min + STV&#8221; sample) is applied, virus at the cell surface can no longer be detected by STV-Cy3 s...

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Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

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JoVE Journal

Immunology and Infection

9.9K Views.  University of Zurich. We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry. 

Video: Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

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 ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

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

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

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 leukocytes.

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.