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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	<li>Edinger, T. O., Pohl, M. O., Stertz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+influenza+A+virus:+host+factors+and+antiviral+targets.">Entry of influenza A virus: host factors and antiviral targets.</a> J Gen Virol. 95, 263-277 (2014).</li><li>Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virolog. 2, (2007).</li><li>Matlin, K. S., Reggio, H., Helenius, A., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+entry+pathway+of+influenza+virus+in+a+canine+kidney+cell+line.">Infectious entry pathway of influenza virus in a canine kidney cell line.</a> J Cell Biol. 91, 601-613 (1981).</li><li>Sieczkarski, S. B., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+virus+can+enter+and+infect+cells+in+the+absence+of+clathrin-mediated+endocytosis.">Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis.</a> J Virol. 76, 10455-10464 (2002).</li><li>Rust, M. J., Lakadamyali, M., Zhang, F., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+endocytic+machinery+around+individual+influenza+viruses+during+viral+entry.">Assembly of endocytic machinery around individual influenza viruses during viral entry.</a> Nat Struct Mol Biol. 11, 567-573 (2004).</li><li>Vries, E., 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=Dissection+of+the+influenza+A+virus+endocytic+routes+reveals+macropinocytosis+as+an+alternative+entry+pathway.">Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway.</a> PLoS Pathog. 7, e1001329 (2011).</li><li>Vries, E., 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=Influenza+A+virus+entry+into+cells+lacking+sialylated+N-glycans.">Influenza A virus entry into cells lacking sialylated N-glycans.</a> Proc Natl Acad Sci USA. 109, 7457-7462 (2012).</li><li>Londrigan, S. 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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 knockdown or overexpression of a protein of interest. Thus, the assay allows for modifications of the protocol depending on the individual needs. Of note, the assay can also be performed for a series of time points to measure virus internalization over time and obtain kinetic information. This might be particularly important if the assay were to be adapted for a different virus with unknown internalization kinetics.
However, it should be noted that the window of detection is relatively small as differences in attachment or internalization occur on a linear scale due to the absence of viral replication. To increase confidence and to reduce noise we recommend including controls (e.g. the controls describe above) and several replicates. While the results shown depict a representative experiment, we observed good consistency across different experiments: For example, for attachment, the percentage of Cy3-positive cells ranged from 60-80%; for internalized virus we obtained values ranging between 40-60%.
Before the assay is started, it is important to titrate the working concentrations of the used reagents such as STV, STV-Cy3 and the amount of biotinylated virus. Please note that the amount of reagents and incubation times can vary depending on the cell line and virus strain used. To maximize the window of detection, the percentage of cells positive for virus attachment should be as high as possible in the &#8220;0 min and &#8220;30 min&#8221; sample (depending on the amount of biotinylated virus used) and as low as possible in the &#8220;0 min +STV&#8221; sample (depending on the amount of STV used). In addition, noise can be reduced by keeping the cells at 4 &#176;C during centrifugation and by washing with ice-cold PBS.

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><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&amp;nbsp;&amp;nbsp;</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>&lt;a href=&quot;https://extranet.fisher.co.uk/insight2_ch/getProduct.do?productCode=W9971E&quot;&gt;W9971E&amp;nbsp;&lt;/a&gt;</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>&amp;nbsp;Lucerna chem&amp;nbsp; 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 staining. As a result, the signal intensity in the &#8220;0 min + STV&#8221; sample is strongly reduced compared to the &#8220;0 min&#8221; sample (Figures 1 and 2). Upon raising the temperature to 37 &#176;C, attached virus is taken up into the cells (&#8220;30 min&#8221; sample). Internalized virus is not sensitive to blocking with unlabeled STV which results in a less dramatic drop in signal intensity in the &#8220;30 min + STV&#8221; samples relative to the &#8220;30 min&#8221; sample in which no STV blocking was applied (Figures 1 and 2).
As controls we employed mock infection, NA treatment and SA treatment. Figure 3 displays the percentages of Cy3-positive cells for all experimental conditions described in section 4. Mock-infected cells give the background signal intensity for the experiment and act as negative control for virus attachment (Figure 4). An additional control for virus attachment is treatment with bacterially expressed NA. NA removes sialic acid, the receptor for IAV attachment, from glycoproteins on the cell surface and thereby reduces binding of the virus to target cells 17. As shown in Figure 4, treatment with NA strongly abrogates IAV attachment by approximately 90% compared to untreated cells. Regarding internalization, we used SA as control. SA treatment promptly depletes cellular ATP levels thereby inhibiting energy consuming processes such as endocytosis 13. Incubation of cells with SA during and after infection inhibited internalization of virus particles. The relative amount of internalized virus (depicted in Figure 5) was raised from approximately 10% to 45% by incubation at 37 &#176;C for 30 min in untreated cells. Following SA treatment however, the percentage of relative internalized virus was around 15% for both, cells incubated for 0 and 30 min at 37 &#176;C. This indicates that indeed, SA reduces the effective uptake of viral particles into cells.
<img alt="Figure 1" src="/files/ftp_upload/53372/53372fig1.jpg" /> 
Figure 1.&#160;Principle of the assay. Cartoon explaining the principle of the attachment/internalization assay. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53372/53372fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53372/53372fig2.jpg" /> 
Figure 2.&#160;Representative result for IAV attachment and internalization. (A) Histogram showing the signal intensity of the Cy3 signal for untreated cells. Shown are the &#8220;0 min&#8221;, &#8220;0 min + STV&#8221;, &#8220;30 min&#8221; and &#8220;30 min + STV&#8221; sample. (B) Percentage of Cy3-positive cells from A. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53372/53372fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53372/53372fig3.jpg" /> 
Figure 3.&#160;Representative result for attachment and internalization including controls. Percentage of Cy3-positive A549 cells following indicated treatment. Shown are mock-infected, untreated, NA-treated and SA-treated cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53372/53372fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53372/53372fig4.jpg" /> 
Figure 4.&#160;Inhibition of virus attachment by neuraminidase. (A) Histogram showing the Cy3 signal intensity of &#8220;0 min&#8221; samples. Displayed are untreated cells (in blue) and two control samples: mock-infected and NA-treated cells (in red and orange, respectively). (B) Percentage of Cy3-positive cells from A. Shown are mock, untreated and NA-treated cells from the &#8220;0 min&#8221; sample. The value for untreated cells was set to 100%. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53372/53372fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53372/53372fig5.jpg" /> 
Figure 5. Inhibition of virus internalization by sodium azide (SA). (A) Histogram showing the Cy3 signal intensity of the &#8220;30 min&#8221; and &#8220;30 min + STV&#8221; samples from untreated (in red and orange, respectively) and SA-treated (in blue and green, respectively) cells. (B) Percentage of relative internalized virus from untreated and SA-treated cells after 0 and 30 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53372/53372fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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Nanyang Technological University

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JoVE Journal

Immunology and Infection

10.0K 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

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Biology

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

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Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging

Source: Yang, J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62764">Automated, High-Throughput Detection of Bacterial Adherence to Host Cells.</a> J. Vis. Exp. (2021)
This video demonstrates the bacterial adherence assay to evaluate GFP-tagged bacterial adherence to epithelial cell culture to study the dynamics of host-pathogen interactions using fluorescence microscopy-based imaging.

1. Bacterial adherence and host cell staining

	First, wash the seeded A549 cell monolayers three times with warm 1x PBS. For each wash, add 100 &#956;L ...

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

Summary

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Chapters in this video

High-throughput Detection Method for Influenza Virus

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

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

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging