Name: Visualizing Dengue Virus through Alexa Fluor Labeling (Video) | JoVE Journal
Uploaded: 2011-07-09T12:00:00.000+00:00
Duration: 9 min 11 s
Description: Learn to fluorescently label dengue virus using Alexa Fluor dye for microscopic visualization of virus-cell interactions. This method enables detailed study of early infection stages in virology research.

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5 minutes

This work has been funded by the National Medical Research Council, Singapore.

1. Alexa Fluor labeling of dengue virus
<ol>
	<li>Before the labeling reaction, purify dengue virus with sucrose cushion and prepare the necessary reagents and equipment as indicated in the protocol.</li>
	<li>Prepare fresh 0.2M sodium bicarbonate buffer, pH 8.5 (labeling buffer), and 1.5M hydroxylamine buffer, pH 8.3 (stop reagent), just before labeling and filter 
	sterilize with 0.2μm syringe filters.</li>
	<li>Dilute approximately 3x108 plaque forming units (pfu) of purified dengue virus in 1ml of labeling buffer in a 2ml tube. This can be scaled up proportionally for batch labeling of virus.</li>
	<li>Reconstitute the lyophilized Alexa Fluor 594 (AF594) succinimidyl esters to 1mM in labeling buffer immediately prior to the labeling reaction. Other fluorochromes from the Alexa Fluor dye series may be used according to one’s needs. Minimize exposure to light from this step onwards.</li>
	<li>Add 100μl of the 1mM AF594 dye to the diluted virus while stirring gently with the pipette tip.</li>
	<li>Incubate the labeling reaction mix at room temperature for 1hr in the dark. Mix by gentle inversions every 15mins.</li>
	<li>Spin the tube briefly in a tabletop centrifuge and add 100μl of stop reagent to the reaction mix while stirring gently with the pipette tip. </li>
	<li>Incubate at room temperature for an additional hour in the dark. Mix by gentle inversions every 15mins.</li>
</ol>
2. Purifying Alexa Fluor labeled dengue virus
<ol>
	<li>In the meantime, equilibrate the purification column with buffer of choice. In this experiment, a PD-10 column is equilibrated with 25ml of HNE buffer (5mM Hepes, 150mM NaCl, 0.1mM EDTA), pH 7.4, before use.</li>
	<li>Apply the labeled virus to the top of the column and start collecting the flow-through once the labeled virus enters the matrix. Fill the column with HNE buffer 
	once all the labeled virus has entered the matrix. Discard the first 2.5ml of flow-through and collect the next 2ml of labeled virus fraction.</li>
	<li>Aliquot and store purified AF594 labeled dengue virus in -80°C, away from light source.</li>
</ol>
<ol>
	<li>Thaw one aliquot and determine the titer of the labeled virus by plaque assay before using the batch of labeled virus.</li>
	<li>Seed 5x104 per well of Vero cells, grown in M-199 growth medium, in a 4-well plate with a coverslip on the bottom of well a day before infection.</li>
	<li>Remove the culture supernatant in well and infect the cells with multiplicity of infection of 1 of the labeled virus in 100μl volume (diluted in M-199 maintenance medium as required) for 10min at 37°C.</li>
	<li>Remove the inoculums and wash the coverslips twice in 1xPBS.</li>
	<li>Fix the cells in 3% paraformalydehyde for 30mins.</li>
	<li>Wash the coverslips 3 times in 1xPBS.</li>
	<li>Permeabilize the cells with permeabilization solution containing 0.1% saponin and 5% BSA in 1xPBS for 30mins.</li>
	<li>Incubate the cells with undiluted centrifugation-clarified supernatant of 3H5 monoclonal antibody hybridoma culture for 1hr in a humid chamber, protected from light.</li>
	<li>Wash the coverslips 3 times in wash buffer (1xPBS containing 1mM calcium chloride, 1mM magnesium chloride and 0.1% saponin).</li>
	<li>Incubate the cells with AF488 anti-mouse IgG antibody, 1:100 diluted in permeabilization solution, for 45mins in humid chamber, protected from light.</li>
	<li>Wash the coverslips 3 times in wash buffer and rinse once in deionized water.</li>
	<li>Dab the edge of coverslip against a paper towel to drain excess water and mount on to glass slide with 8μl Mowiol 4-88 containing 2.5% Dabco.</li>
	<li>Allow the mounting solution to set overnight at 4°C before viewing using a Zeiss confocal microscope. Sequential acquisitions should be performed exciting one fluorophore at a time and switching between the detectors concomitantly.</li>
	<li>Images are then analyzed for co-localization of the E protein antibody staining with the labeled virus using the Zeiss LSM Zen software to estimate the degree of labeling.</li>
</ol>
3. Representative Results
An example of the yield of dengue virus labeled with AF594 dye is shown in Figure 2. Normally, less than 10-fold drop from the initial titer should be observed following successful labeling. However, it should be noted that all buffers have to be prepared fresh for the labeling to be successful and the Alexa Fluor succinimidyl esters should be used immediately upon reconstitution as they hydrolyze into nonreactive free acids in aqueous solutions8.
Next, the labeled virus has to be checked for sufficient fluorescence before use in experiments. A simple immunofluorescence assay was done on Vero cells and the degree of labeling can be estimated from the co-localization of the labeled virus with anti-E protein antibody staining. Several cells were examined and a typical confocal image is shown in Figure 3. Co-localization analysis of the images using the LSM Zen software demonstrated overlap coefficients ranging from 0.65 to 0.8, suggesting that approximately 65 to 80% of the virions were labeled with the dye.
<img src="/files/ftp_upload/3168/3168fig1.jpg" alt="Dengue virus labeling process; includes Alexa Fluor labeling, fluorescence assay and microscopy." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1.Overall scheme depicting the Alexa Fluor dye labeling of dengue virus procedure. First, the relevant buffers and purified dengue virus are prepared. The Alexa Fluor dye is reconstituted and added to the dengue virus diluted in labeling buffer. The reaction is then stopped 1 hour later with the addition of stop reagent. Subsequently, the labeled virus is purified through a size exclusion column to remove free dye. Finally, the labeled virus is re-titrated by plaque assay and tested for fluorescence.
<img src="/files/ftp_upload/3168/3168fig2.jpg" alt="Virus titer analysis; bar chart; before vs. after treatment; titer pfu/ml; compare viral load." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Mean number of viable virions (pfu/ml) as determined on a plaque assay before and after AF594 labeling. An aliquot of the AF594 labeled dengue virus is thawed and re-titrated by plaque assay and it typically shows less than 10-fold drop from the starting titer. Error bars indicate standard deviation of duplicates.
<img src="/files/ftp_upload/3168/3168fig3.jpg" alt="Fluorescence microscopy of E protein with AF594 labeling; three-panel diagram showing protein localization." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Co-localization of AF594 labels with dengue virus E proteins in Vero cells. cells grown on coverslips the day prior were infected with AF594 labeled dengue at MOI of 1 for 10 minutes at 37°C. The cells were subsequently fixed and labeled with anti-E antibody, and examined for colocalization of E protein (green) and AF594 labeling (red). Fluorescent signals were visualized under 63X magnification using Zeiss LSM 710 confocal microscope. Scale bar is 10μm. Yellow indicate areas of colocalization, as shown in the inset.

<ol><li>Olenych, S. G., Claxton, N. S., Ottenberg, G. K. &amp, Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Fluorescent+Protein+Color+Palette%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">The Fluorescent Protein Color Palette.</a> Current Protocols in Cell Biology. 21.5, 1-34 (2007).</li>
<li>Panchuk-Voloshina, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alexa+dyes%2C+a+series+of+new+fluorescent+dyes+that+yield+exceptionally+bright%2C+photostable+conjugates%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.</a> J Histochem Cytochem. 47, 1179-1188 (1999).</li>
<li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+fluorescent+protein+technology%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li>
<li>Lippincott-Schwartz, J., Patterson, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+use+of+fluorescent+protein+markers+in+living+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Development and use of fluorescent protein markers in living cells.</a> Science. 300, 87-91 (2003).</li>
<li>Kuhn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+dengue+virus%3A+implications+for+flavivirus+organization%2C+maturation%2C+and+fusion%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Structure of dengue virus: implications for flavivirus organization, maturation, and fusion.</a> Cell. 108, 717-725 (2002).</li>
<li>Aguilar, J. S., Roy, D., Ghazal, P., Wagner, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dimethyl+sulfoxide+blocks+herpes+simplex+virus-1+productive+infection+in+vitro+acting+at+different+stages+with+positive+cooperativity.+Application+of+micro-array+analysis%5BTitle%5D)+AND+%22BMC+Infect+Dis%22%5BJournal%5D)">Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis.</a> BMC Infect Dis. 2, 9-9 (2002).</li>
<li>Zhang, S. L., Tan, H. C., Hanson, B. J., Ooi, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+method+for+Alexa+Fluor+dye+labelling+of+dengue+virus%5BTitle%5D)+AND+%22J+Virol+Methods%22%5BJournal%5D)">A simple method for Alexa Fluor dye labelling of dengue virus.</a> J Virol Methods. 167, 172-177 (2010).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Alexa+Fluor+Succinimidyl+Esters+%28Molecular+Probes%22">Alexa Fluor Succinimidyl Esters (Molecular Probes</a>. , Invitrogen. (2009).</li>
<li>Huang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+proteins+stained+by+Alexa+dyes%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Analysis of proteins stained by Alexa dyes.</a> Electrophoresis. 25, 779-784 (2004).</li>
<li>Freistadt, M. S., Eberle, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+poliovirus+for+flow+cytometric+cell+surface+binding+studies%5BTitle%5D)+AND+%22J+Virol+Methods%22%5BJournal%5D)">Fluorescent poliovirus for flow cytometric cell surface binding studies.</a> J Virol Methods. 134, 1-7 (2006).</li>
</ol>

We have nothing to disclose.

Although AF594 dye was used in this report, a wide range of fluorophores in the Alexa Fluor succinimidyl esters series is available with similar labeling chemistry. This could extend the labeling application beyond imaging. Flow cytometry can be used as an alternative to confocal microscopy for estimating the degree of labeling for fluorophores that can be excited and detected by the FACS machine.
Alexa Fluor dyes are small molecules that react with free amino groups, primarily arginine and ly...

<table><tbody><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td></td></tr><tr><td>Hydroxylamine</td><td>Sigma-Aldrich</td><td>159417</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck &amp;amp; Co., Inc.</td><td>106498</td><td></td></tr><tr><td>AF594 succinimidyl esters</td><td>Molecular Probes, Life Technologies</td><td>A20004</td><td></td></tr><tr><td>PD-10 column</td><td>GE Healthcare</td><td>17-0851-01</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H6147</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td></td></tr><tr><td>M-199</td><td>Invitrogen</td><td>11150</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>4-well plate</td><td>Nalge Nunc international</td><td>176740</td><td></td></tr><tr><td>Coverslips</td><td>Einst</td><td>0111520</td><td></td></tr><tr><td>Microscope slide</td><td>Sail Brand</td><td>7105</td><td></td></tr><tr><td>3H5 hybridoma</td><td>ATCC</td><td>HB46</td><td></td></tr><tr><td>10x PBS</td><td></td><td>BUF-2040-10X1L</td><td>1st Base</td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>S4521</td><td></td></tr><tr><td>BSA</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>Paraformaldehye</td><td>Sigma-Aldrich</td><td>15,812-7</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Calbiochem</td><td>475904</td><td></td></tr><tr><td>Dabco</td><td>Sigma-Aldrich</td><td>D27802</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Confocal microscope</td><td>Carl Zeiss, Inc.</td><td>LSM 710</td><td></td></tr><tr><td colspan="4">To prepare M-199 growth medium, add 50ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter. </td></tr><tr><td colspan="4">To prepare M-199 maintenance medium, add 15ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter.</td></tr></tbody></table>

visualizing dengue virus through alexa fluor labeling

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

The overall objective of this experiment is to Fluorescently label dengue virus for microscopic studies. Prepare necessary Buffers and purify dengue virus with sucrose cushion reconstitute the Amy React Alexa flow dye and dengue virus in labeling buffer. Label the dengue virus with a hundred micromolar of Alexa flow.Die for one hour and stop the labeling reaction with the sub reagent. Purify the label dengue virus through a size exclusion column. Retitrate the labeled virus by plaque assay and estimate the degree of labeling by immunofluorescence assay.Hi, I'm Sam Jang from the DSO National Laboratories working in Dr.Wee's lab in the program of emerging infectious diseases after NUS graduate medical school. Today I'm gonna show you how to fluorescently labeled Denki virus with Amy Reactive Alexa flu dye for imaging studies. Alexa, flu die, have superior for the stability and are less pH sensitive than common diet such as fluorescein and rumine task, making them ideal for studies on cellular uptake and endosomal transport of the virus.Alexa flaw labeled dengue virus will facilitate the understanding of dengue pathogenesis. So let's get started. Before the labeling reaction, prepare fresh labeling buffer and stop reagent according to the written protocol and purify dengue virus by sucrose cushion, add appropriate volume of the purified dengue virus to labeling buffer in a micro centrifuge tube to achieve a concentration of 300 million forming units per milliliter.This can be scaled up proportionately. For batch labeling of virus. Reconstitute the Lyophilized Alexa floor dye to one millimolar in labeling buffer just prior to the labeling reaction.Minimize exposure to light from this step onwards. For demonstration purpose, the Alexa floor dye will be left unprotected from light 800 microliters of the one millimolar. Alexa flow die to the diluted dengue virus while stirring gently with the micro Pipet tip.Incubate for one hour at room temperature protected from light mixed by gentle inversions every 15 minutes after one hour, Centrifuge the tubes briefly. An 800 microliter of the sub region while staring gently with the micro pipette incubate for an additional one hour at room temperature. Protected from light makes by gentle inversions every 15 minutes.In the meantime, it equate the purification column with 25 milliliters of HNE buffer. After the one hour Incubation, apply the labeled virus to the column and start collecting the flow through. Fill the column with HNE buffer.Once All the labeled virus has entered the matrix, discard the first 2.5 milliliters of flow through and collect the next two milliliters of the labeled virus fraction. Ecor and saw the purified labeled virus at Minus 80 degrees Celsius. Determine the title Of the labeled virus by plaque assay.In a typical experiment, less than tenfold drop in the virus title should be observed. Post labeling, an immunofluorescence assay should be carried out to evaluate the degree of labeling. To do that enumerate viral cells using a hemo cytometer and appropriate volume of cells to growth media to achieve a density of 50, 000 cells per milliliter.See one milliliter of viral cells per well in a four well plate containing a sterile glass cover.Sleep. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide. Remove the plate from the incubator and aspirate the supinator from well in fact, the cells with approximately one MOI of the labeled virus in a hundred microliters volume for 10 minutes at 37 degrees Celsius.Remove the Inoculum and wash the cells with 0.5 milliliters of PBS buffer per well. Spirit dis and repeat this procedure two more times. Fix the cells with 200 microliters of 3%per for multi height per well at room temperature for 30 Minutes.Wash the cells three times with 0.5 milliliters of PBS buffer per well. Alize the cells with ization solution at room temperature for 30 Minutes. Remove the cover sleep from well using fine forceps.Drain excess fluid by DPing the edge of the cover. Slip against a paper white. Place the cell side of the cover.Sleep onto 25 microliters of dengue e protein antibody on a piece of parfum. Incubate for one hour in a humid chamber at room temperature protected from light. Next, transfer the cover slip to a six well plate and add two milliliters of wash buffer per oil.Aspirate the supinate. Repeat these washing Steps two more times after the last wash. Remove the cover slip from well using fine fp.Drain the excess fluid by dipping the edge of the cover. Slip against a paper wipe. Place the cell side of the cover.Sleep onto 25 microliters of Alexa floor for eight eight anti mouse secondary antibody on a piece of perfume. Incubate for 45 minutes in a humid chamber at room temperature protected from light after the incubation is completed, transfer the cover sleep to a six Well plate and wash the cells three times with two milliliters of wash buffer For each wash after the last Wash. Remove the cover slip from the well and rinse once in the ionized water.Drain the excess fluid by dipping against a paper. Wipe mount the cover slip onto a microscope light with eight microliters of mild mounting solution. Allow the mounting solution to set overnight air four degrees Celsius.The sample is now ready to be visualized with a fluorescent or confocal microscope. Place a small drop of immersion oil on the surface of the cover sleep and fasten the microscope. Slide to the microscope stage.Adjust the stage until the microscope objective comes into contact with the immersion oil. For a claral image, turn off the ambient lights using the Zes Zen software. Click on the ocular tap for direct observation of the sample through the eyepiece of the microscope.Find a fill with cells and adjust the focus. Now switch to the acquisition mode. To minimize crosstalk between floral falls, select separate tracks for Alexa floor 4 8 8 and finite four.Dyes and set acquisitions to sequential excitation and detection. Adjust the laser power gain and offset to ensure that the image is not over or under exposed. Capture the image and analyze an area of interest using colocalization function.The overlap coefficient can be used to estimate the degree of labeling of dengue virus by the Alexa Flo Dye. A simple immunofluorescence assay was done on viral cells and the degree of labeling can be estimated from the colocalization of the labeled virus With anti e protein, anti antibody staining overlap coefficience range from 0.65 to 0.8 suggesting that approximately 65 to 80%of the variances were labeled with the Alexa floor dye. For the labeling procedure to be successful, it is important to prepare the labeling buffer and start raging fresh and reconstitute the Alexa floor digest prior to the labeling.The concentration of dye to use can be further optimized to suit your needs. This method of the horizon labeling dengue virus can potentially be adapted to label other viruses. So that's it.Thanks for watching and good luck for your labeling experiment.

visualizing-dengue-virus-through-alexa-fluor-labeling

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Learn to fluorescently label dengue virus using Alexa Fluor dye for microscopic visualization of virus-cell interactions. This method enables detailed study of early infection stages in virology research.

Visualizing Dengue Virus through Alexa Fluor Labeling

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