Name: Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy (Video) | JoVE Journal
Uploaded: 2010-10-07T17:00:00.000+00:00
Duration: 13 min 8 s
Description: Learn live confocal microscopy for visualizing HIV transfer across virological synapses between infected and uninfected T cells. This protocol enables real-time observation of viral transmission dynamics.

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5 minutes

This review was supported by National Institutes of Health grants, AI074420-02, Burroughs Wellcome Investigator Award, Hirschl Career Scientist Awards to B.K.C. and by the NSF Center for Biophotonics Science and Technology, Cooperative Agreement PHY012099, and a UCD Health System Research Award to T.H. UCD CTSC NCRR ULRR024146.

This method was used in the research reported in <a href="http://www.sciencemag.org/cgi/content/abstract/323/5922/1743">H&uuml;bner et al. Science 323: 1743-1747 (2009)</a>.
1. Overview
Cell-to-cell spread of HIV was originally described using HIV infected Jurkat cells as donor cells and primary CD4+ T lymphocytes as acceptor cells 1,2,3. In these studies, virus was detected in fixed cells using antibody staining. To track the transfer of virus from cell to cell in live cells, we exploit a recombinant, infectious molecular clone of HIV called HIV Gag-iGFP 4. It carries the green fluorescent protein (GFP) inserted internally into the Gag protein between the MA and CA domains. This molecular clone of HIV retains infectivity without the need for helper virus. Virus particles made from this clone are stoichiometrically loaded with green fluorescent protein, providing a strong fluorescence signal to monitor viral assembly and transfer between cells. When used in conjunction with inert cell-tracking fluorescent dyes, recipient cells can be discriminated from input donor cells, and allowing one to visualize the transfer of HIV from an infected T cell to an uninfected T cell 5. Virological synapse formation and transfer of virus from one cell to another can then be observed in living cells while they are cultured in a sealed, gas-permeable imaging chamber.
(Day 1)
2. Preparation of HIV Gag-iGFP Transfected Jurkat T Cells
<ol>
	<li>Prepare the human CD4+ T cell line Jurkat (ATCC, Manassas, VA) by carefully maintaining the cells at a concentration between 2 x 105 and 8 x 105 cells/mL in Jurkat culture media (RPMI 1640, 10% fetal bovine serum, 100 units/mL penicillin, and 100 &mu;g/mL streptomycin). Key to success: Culture of the Jurkat cells at concentrations in excess of 8 x 105 cells/mL will result in a reduction in transfection efficiency. 
Note: The transfection of HIV Gag-iGFP proviral DNA into T cells, as described below, results in the production of a replication-competent human immunodeficiency virus. As such, all procedures are to be undertaken only by trained laboratory personnel in certified biosafety level 2+ or biosafety level 3 (BL2+/BL3) tissue culture rooms. Work with infectious HIV in imaging facilities must be approved by your local institutional biosafety office.</li>
	<li>Transfect the Jurkats with the viral plasmid, HIV Gag-iGFP using the Amaxa nucleofection method (Lonza, Walkersville, MD). Pellet 5 x 106 cells by centrifugation for 10 min at 150 x g. Aspirate supernatant and wash the cells in sterile phosphate buffered saline (PBS). Centrifuge the cells and resuspend the pellet to 97 &mu;L of pre-warmed, nucleofector solution V containing the manufacturer's supplement. Add three &mu;L of endotoxin free HIV Gag-iGFP plasmid (1 &mu;g/&mu;L) to the cell suspension and mix gently. Transfer the cell suspension to a cuvette and nucleofect using program S-18. Immediately transfer the cells to 3 mL of prewarmed Jurkat culture media without antibiotics.</li>
	<li>To prepare CD4+ target cells for virological synapses, we obtain human peripheral blood mononuclear cells from buffy coats by using a standard Ficoll-Paque (GE Healthcare, Uppsala, Sweden) protocol. CD4+ T cells are then negatively selected using a magnetic bead isolation kit (Miltenyi Biotec, Auburn, CA). These may be cryogenically stored in liquid N2 in aliquots of 5 x 106 cells/500 &mu;L of freezing media (90% fetal bovine serum/10% DMSO). Thawed primary CD4+ T cells are cultured in RPMI 10% FBS, supplemented with 10 units/mL of IL-2.</li>
</ol>
(Day 2)
3. Recovery of Live Cells from Jurkats Transfected with HIV Gag-iGFP and Staining of Target Cells with Fluorescent Dyes
<ol>
	<li>Allow the Jurkat cells to recover from the transfection overnight. Then remove the cellular debris by centrifugation over a ficoll hypaque density gradient material. Dispense 1.5 mL of Ficoll-Paque to the bottom of a 15 mL conical tube. Gently overlay this gradient material with the transfected Jurkats in a 5 mL volume of RPMI. Centrifuge the cells at 400 x g for 20 min at room temperature with the brake off. Carefully remove the cells from the media/Ficoll interface using a pipette. Transfer them to a new 15 mL conical tube. Dilute the cells with RMPI to a total volume of 15 mL and centrifuge at 300 x g for 10 min. Resuspend the pellet in 3 mL of Jurkat culture media in a 2 cm well (6-well plate) and return the cells to the tissue culture incubator.</li>
	<li>To distinguish target cells from donor cells in cell-cell transfer experiments we prelabel the CD4+ target cells with fluorescent dyes. Primary CD4+ T cells are labeled the evening before use. We have had excellent success labeling T cells with both CellTracker and CellTrace dyes (Invitrogen, Carlsbad, CA). While the dye concentration and incubation times must be optimized depending on the particular dye and cell type used, a representative protocol follows. Pellet 4 x 106 primary CD4+ T cells by spinning at 400 x g for 10 min. Wash the cells with 5 mL of PBS and resuspend in 2 mL of PBS. Add CellTracker Orange (CMTMR, Invitrogen, Carlsbad, CA) to a final concentration of 1.5 &mu;M and incubate at 37&deg;C for 20 min. Add 8 mL of complete Jurkat media and centrifuge at 400 x g for 10 min. Resuspend the cells in 3 mL of complete media supplemented with 10 units/mL of IL-2 and place in the tissue culture incubator overnight.</li>
</ol>
(Day 3)
4. Monitoring Cell-to-cell Transfer of HIV Gag-iGFP using Live Cell Imaging
We routinely use HIV Gag-iGFP transfected Jurkat cells 48 hours post-transfection. However, we have successfully used cells as early as 24 hours, and as late as 72 hours post-transfection.
<ol>
	<li>Approximately 48 hours post-transfection, HIV Gag-iGFP expressing Jurkats are counted, washed with CO2-independent media (Invitrogen, Carlsbad, CA), and resuspended in live cell imaging media (CO2 independent media, 10% Fetal Bovine Serum, 100 U/mL penicillin, and 100 &mu;g/mL streptomycin, 10 units/mL IL-2) at a concentration of 1-3 x 107 cells/mL.</li>
	<li>In a similar fashion, wash and resuspend labeled primary CD4+ T cells at a concentration of 1-3 x 107 cells/mL in live cell imaging media.</li>
	<li>Mix HIV Gag-iGFP-transfected Jurkats with primary CD4+ T cells at a 1:2 or 1:3 ratio and load into a tissue culture treated, gas permeable, microchamber (Ibidi, Verona, WI). For example, mix 20 &mu;L of HIV Gag-iGFP transfected Jurkats with 40 &mu;L of labeled primary CD4+ T cells. Load 50 &mu;L of this mixture into an Ibidi chamber and seal the chamber with plugs. Secure the plugs by wrapping the plug/Ibidi chamber interface with parafilm.</li>
</ol>
5. Considerations for microscopy platforms: spinning disk confocal imaging
After loading an Ibidi imaging chamber with cells, we immediately mount the device onto an inverted optical microscope (iX71 Olympus, Center Valley, PA). The Ibidi chamber should be transferred to the microscope only by BL2+-trained laboratory personnel wearing appropriate safety garments. Although live-cell imaging commonly uses expensive incubation chambers to maintain a stable 37&deg;C environment, this was achieved using a simple and economical thermostatic heater (ASI 400, Nevtek, Williamsville, VA) along with a T-type thermocouple tip mounted next to the sample for proper temperature feedback. We have found using CO2-independent media allows us to maintain high cell viability while avoiding the use of a CO2 chamber. To buffer the platform against temperature fluctuations in the room and to block out background room light, a large black shroud is draped over the whole microscope/heater setup creating an insulated air pocket around the system. This greatly reduces temperature variation and associated temperature-related sample drift, but requires pre-heating for 30 minutes prior to mounting the sample.
6. Data Acquisition, Transfer and Image Processing
Images are taken using a 60x 1.42 NA oil immersion objective (UPlanApo N, Olympus) as the high numerical aperture (NA) greatly improves resolution. To create 3D confocal images, scanning is performed by a spinning disk confocal unit (CSU-10, Yokagawa, Japan) that simultaneously uses &gt;800 confocal spots to dramatically increase the scan rate. To compliment its speed, the CSU-10 is paired with a highly sensitive electron-multiplying charged-coupled device (EMCCD, iXon+ 897, Andor Technologies, Ireland) camera to allow fast, low light imaging which reduces both photobleaching and phototoxicity. The fluorescence light source is a multi-wavelength ArKr ion gas laser (Innova 70C, Coherent, Santa Clara) where the desired wavelength (488 nm) and laser power measured right before the microscope objective (&lt;1 mW) are selected by an acousto-optic tunable filter (AOTF), (Andor Technologies). To further reduce photobleaching and extend total imaging time, the AOTF rapidly (microseconds) shuts off the laser beam every time the camera is inactive (10-20 ms) while the newly acquired image is delivered from the CCD (15-30 ms exposure) to the computer. The laser light is coupled into the spinning disk system using a 405/488/568/647 quad band dichroic mirror (Semrock, Rochester, NY). Fluorescence emission is filtered using a 525/50 bandpass filter (Semrock) inside a filter wheel (Ludl Electronic Products, Hawthorne, NY) mounted between the EMCCD camera and the confocal spinning disk unit. All hardware and acquisition, including a z-stage (Mad City Labs, Madison, WI), are controlled by the Andor iQ v1.8 software. To maximize the speed of acquisition at around 1.2-1.9 second 3D image stacks, we crop the recording region of the EMCCD's camera down to the cell-pair's immediate area. Furthermore, at the expense of some information in z, a large z-step between 0.45-0.75 &mu;m can be used to speed up acquisition. Imaging under these conditions can last up to 6 hours - using continuous 20-60 minute segments - with minimal photobleaching. Altogether, each data segment typically occupies 5-20 Gb of space as tens of thousands of images are taken. To facilitate the transport and analysis of the large data files, each acquisition set needs to be broken down and exported as 1 Gb TIFF image file segments using the Andor iQ v1.8 software. From here, many excellent software packages are available for image analysis, such as Metamorph, Volocity and ImageJ (we recommend its variation Fiji).
When two fluorescent markers are to be tracked, we simultaneously record both of them without slowing down acquisition by using a "split screen mode." Both markers are excited by two different ArKr laser lines at once (often 488 and 568 nm). Inserted directly before the EMCCD camera is an image splitter (OptoSplit II, Cairn, Kent, UK) that separates the two different images. Each image is then independently filtered using fluorescence filters (Semrock) and projected side-by-side on the camera at once creating the "split-screen" effect. This would then require manual cropping and aligning of images later in image processing.
7. Considerations for Image Processing and Data Analysis
We typically perform image analysis with Volocity (PerkinElmer, Waltham, MA) on Macintosh computers (Apple, Inc). Spinning disk laser confocal microscopy images are first adjusted in their intensity to correct for photobleaching, then deconvolved with Volocity. Intensity measurements and tracking of Gag puncta is performed with the Volocity Quantification module. For image sets where the movement relative to a pre-formed synapse needs to be calculated, an automated tracking algorithm is employed which tracks the synaptic button throughout the entire sequence. Manual inspection of the regions of interest defined by the autotracking software must be performed to confirm that the software has correctly tracked the desired object. For objects where the contrast with the surrounding objects is too weak, manual tracking may be performed on a frame-by-frame basis. The Volocity software package allows the export of XYZ location, volume and integrated signal within the desired objects. The distance from the synapse and velocity of the tracked object are calculated by normalizing movements to the center of the synaptic button.
8. Representative Results
Within 10 to 15 minutes of mixing one should be able to visualize HIV Gag-iGFP expressing Jurkat cells adhering to primary CD4+ T cells. Imaging these conjugates, one can assess the movements of Gag puncta in infected cells as well as in target cells after synaspe. One may observe the movement of Gag-iGFP puncta into target cells soon after synapses are formed.

<ol><li>Blanco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+level+of+coreceptor-independent+HIV+transfer+induced+by+contacts+between+primary+CD4+T+cells%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells.</a> J Biol Chem. 279, 51305-51314 (2004).</li>
<li>Sourisseau, M., Sol-Foulon, N., Porrot, F., Blanchet, F., Schwartz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inefficient+human+immunodeficiency+virus+replication+in+mobile+lymphocytes%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Inefficient human immunodeficiency virus replication in mobile lymphocytes.</a> J Virol. 81, 1000-1012 (2007).</li>
<li>Jolly, C., Kashefi, K., Hollinshead, M., Sattentau, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+cell+to+cell+transfer+across+an+Env-induced%2C+actin-dependent+synapse%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.</a> J Exp Med. 199, 283-293 (2004).</li>
<li>Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequence+of+human+immunodeficiency+virus+type+1+%28HIV-1%29+Gag+localization+and+oligomerization+monitored+with+live+confocal+imaging+of+a+replication-competent%2C+fluorescently+tagged+HIV-1%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.</a> J Virol. 81, 12596-12607 (2007).</li>
<li>Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+3D+video+microscopy+of+HIV+transfer+across+T+cell+virological+synapses%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Quantitative 3D video microscopy of HIV transfer across T cell virological synapses.</a> Science. 323, 1743-1747 (2009).</li>
</ol>

No conflicts of interest declared.

We describe here a simple method for visualizing the transfer of HIV from cell-cell. Recent research from our laboratory and others' suggests that this may be the predominant mode of HIV spread between T cells. The method described here employs a recombinant form of HIV, called HIV Gag-iGFP, which carries a genetically encoded green fluorescent protein tag in the core structural protein, Gag. Because of the high levels of GFP produced in each virus particle, this replication-competent clone of HIV enables researchers to track individual virus particles in real time. This virus should be especially useful in studies concerning the assembly and cell-cell transfer of HIV.
The transfer of HIV by cell-cell transfer is highly efficient. During a three hour co-culture, we routine see virus transferred to 20-30% of primary T cells as assessed by flow cytometry. Similar efficiencies are qualitatively seen during live cell imaging. Virally infected T cells begin forming synapses with uninfected primary T cells almost immediately after the initiation of co-culture. Interesting, not all primary T cells are capable of interacting with HIV-infected Jurkat cells despite forced interactions with laser tweezers (unpublished observations, GM). HIV Gag-iGFP will undoubtedly be useful for determining T cell subsets that are most susceptible to cell-cell transfer, both in vitro and in vivo.

<table><tbody><tr><td>Jurkat T Cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>Leukocyte buffy coats from whole blood</td><td>New York Blood Center</td><td></td><td></td></tr><tr><td>RPMI</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td></td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Nucleofector System and Solution V</td><td>Lonza Inc.</td><td></td><td></td></tr><tr><td>EndoFree Maxi Prep Kits</td><td>Qiagen</td><td></td><td></td></tr><tr><td>HIV Gag-iGFP plasmid</td><td>Chen Laboratory</td><td></td><td></td></tr><tr><td>CD4+ T Cell Isolation Kit</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Celltracker/CellTrace Dyes</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-Independent Media</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Imaging Chambers</td><td>Ibidi</td><td></td><td></td></tr><tr><td>Olympus IX-71 Inverted Microscope</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>PlanApo N 1.42NA 60X oil objective</td><td>Olympus Corporation</td><td></td><td></td></tr><tr><td>Innova 70C ArKr ion gas laser</td><td>Coherent Inc.</td><td></td><td></td></tr><tr><td>CSU-10 Nipkow-type spinning disk confocal unit</td><td>Yokogawa</td><td></td><td></td></tr><tr><td>Di01-T405/488/568/647-13x15x0.5 dichroic beamsplitter</td><td>Semrock</td><td></td><td></td></tr><tr><td>525/50 bandpass filter</td><td>Semrock</td><td></td><td></td></tr><tr><td>iXon+ 897 electron-multiplied charged coupled devise camera</td><td>Andor</td><td></td><td></td></tr><tr><td>ASI 400 Thermostatic Heater</td><td>Nevtek</td><td></td><td></td></tr></tbody></table>

visualizing cell-to-cell transfer of hiv using fluorescent clones of hiv and live confocal microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

This procedure uses live cell imaging to visualize the transfer of GFP labeled HIV across virological synapses between HIV expressing jerk CAT T cells and uninfected primary T cells. The cell to cell transfer assay starts with transecting jerk CAT T cells with HIV gag IG FP, an infectious fluorescent clone of HIV one, the transfected jerk cat cells expressing HIV gag. IG FP are then mixed with C-M-T-M-R labeled uninfected primary T cells and loaded into an imaging chamber where the transfer of GFP labeled HIV across the virological synapse can be observed.The resulting data are analyzed using image analysis software to reveal the details of HIV transfer across T-cell virological synapses. Hi, my name's Benjamin Dale from the laboratory of Ben Chen in the Department of Medicine at the Mount Sinai School of Medicine. My name is Greg McNerney under laboratory of Dr.Thomas Huser at the NSF Center for Biophotonics at the uc Davis Medical Center.And I'm Deanna Thompson, also from the lab of Dr.Thomas Huser. Today we'll show you a procedure for visualizing cell cell transfer of HIV one using live confocal microscopy. In this procedure, jerk hat cells are transfected with an infectious molecular clone of HIV only trained laboratory personnel working in certified biosafety.Level two plus or biosafety level three tissue culture rooms can carry out the protocol shown here. Working with infectious HIV in imaging facilities must be approved by the local institutional biosafety office. Maintain the jerk cat cells to be used as donor cells at a concentration between two times 10 to the fifth and eight times 10 to the fifth cells per milliliter.In jerk cat culture, media cell concentration in excess of eight times 10 to the fifth cells per milliliter results in a reduction in trans efficiency. To begin the procedure, transfect five times 10 to the six jerk cat cells with the viral plasmid, HIV gag IGFP Using the amax nucleo affection method as described in the accompanying written protocol, allow the cells to recover overnight. Peripheral blood mononuclear cells are purified from buffy coats of whole human blood by FICO density gradient centrifugation.The primary CD four positive T cells are purified from the peripheral blood mononuclear cells by negative selection using a CD four positive T cell isolation kit. The negatively selected CD four positive T cells are collected from a magnetic column and cultured in our PMI with 10%FPS that is supplemented with 10 units per milliliter of recombinant human IL two one can also aliquot the isolated primary CD four positive T cells and freeze them with the aid of cryogenic freezing containers. The freezing containers are placed in a minus 70 degree Celsius freezer for a minimum of three to four hours.The cells are then transferred to liquid nitrogen for long-term storage. Now that the jerk head cells are transfected and the target primary CD four positive T cells are isolated, proceed to cleanup and labeling of the cells on the day following the transfection. The jerk cells are now expressing the infectious fluorescent HIV construct.When handling infectious samples, we wear a front closing lab coat and two pairs of gloves. Remove the cellular debris from the transfected cells by centrifugation over a fial high pack density medium. First dispense 1.5 milliliters of fial pack at the bottom of a 15 milliliter conical tube.Then gently overlay the gradient material with the transfected jerk hat cells in a five milliliter volume of our PMI medium. At this point the transfected jerk hat cells are capable of producing infectious HIV. So all pipette tips that come into contact with the HIV expressing cells must be disinfected with 10%bleach solution.Before discarding into a container within the biosafety cabinet, cap the tube and spray the outside of the tube with a solution of 70%ethanol and wipe off with a dry kim wipe centrifuge the cells at 400 Gs for 20 minutes at room temperature with the break off. After centrifugation, use a pipette to carefully remove the cells from the media fial interface and transfer the cells to a new 15 milliliter conical tube. Dilute the cells with our PMI medium to a total volume of 15 milliliters and centrifuge at 300 GS for 10 minutes.At room temperature we suspend the pellet in three milliliters of jerk hat culture media and transfer to a two centimeter well of a six well plate. Place the plate in the tissue culture incubator until ready to use the following day. On the evening of the same day, label the primary CD four positive T cells used as target cells in order to distinguish them from the donor cells First pellet four times 10 of the six primary CD four positive T cells by spinning at 400 Gs for 10 minutes.Then wash the cells with five milliliters of PBS and Resus. Suspend in two milliliters of PBS add cell tracker orange to a final concentration of 1.5 micromolar and incubate at 37 degrees Celsius for minutes. When the cells have finished incubating, add eight milliliters of complete chica media and centrifuge at 400 Gs for 10 minutes.Re suspend the cells in three milliliters of complete media supplemented with 10 units per milliliter of IL two and place in the tissue culture incubator overnight. Following the overnight incubation, the donor and target cells are ready for the cell to cell transfer experiment. Before setting up the cell to cell transfer experiment, prepare the imaging chamber to buffer the platform against temperature fluctuations in the room.And to block out background room light, a large black shroud is draped over the whole microscope heat setup, creating an insulated air pocket around the system. The imaging chamber is maintained at a stable 37 degrees Celsius using a thermostatic heater with a T type thermocouple tip placed next to the sample for temperature feedback. Preheat for 30 minutes prior to mounting the sample.Spinning disc confocal imaging with an inverted optical microscope is used to create 3D confocal images of cell to cell transfer of HIV. All hardware and acquisition including a Z stage, are controlled by the and or IQ version 1.8 software scanning is performed by a CS U 10 spinning disc confocal unit that simultaneously uses more than 800 confocal spots to dramatically increase the scan rate to compliment its speed. The CS U 10 is paired with a highly sensitive electron multiplying charred coupled device camera to allow fast low light imaging, which reduces both photobleaching and phototoxicity.The fluorescence light source is a multi wavelength argon krypton ion gas laser. An and or Oko optic tunable filter or A OTF is used to select the desired wavelengths, which are 488 nanometers and 568 nanometers here while controlling their power to further reduce photobleaching and to extend imaging time. The A OTF rapidly shutters the laser following each 15 to 30 millisecond exposure when the camera is not acquiring images, avoiding unnecessary exposure while the images are transferred from the E-M-C-C-D camera to the computer.The laser light is incorporated into the spinning disc system using a 4 0 5 4 88 5 68 6 47 quad band Dichroic mirror fluorescent emission is separated by wavelength using a 560 nanometer long past diic mirror with an image splitter. The green channel is filtered using a 525 over 50 nanometer band pass filter and the red channel is filtered using a 609 over 54 nanometer band pass filter. The two channels are projected side by side onto the E-M-C-C-D camera.While the imaging chamber is warming up, prepare the cells for the cell to cell transfer experiment. Use a hemo cytometer to count the HIV gag IGFP expressing ette cells. Place the hemo cytometer in a Petri dish to avoid direct exposure to the virus expressing cells.Wash the cells with carbon dioxide, independent media and resuspend in live cell imaging media at a concentration of one to three times 10 of the seven cells per milliliter. The use of carbon dioxide independent media eliminates the need for a carbon dioxide environmental chamber. For imaging.The cells also count, wash and resuspend the labeled primary CD four positive T cells as just shown for the jerk hat cells. Next, initiate cell to cell transfer mix 20 microliters of HIV gag IGFP transfected dur hat cells with 40 microliters of labeled primary CD four positive T cells and load 50 microliters into a tissue culture treated gas permeable. EBD micro chamber seal the chamber with plugs and secure the plugs by wrapping the plug EBIT D chamber interface with paraform.Wipe down the imaging chamber with a chem wipe wedded with 70%ethanol and allow it to air dry briefly in the hood. Transport the sealed imaging chamber in a sealed container to avoid accidental exposure. Should the chamber be dropped in transit immediately after loading the IBD imaging chamber with cells, mount the device onto the microscope.Take images of cell pairs using a 60 x 1.42 NA oil immersion objective As the high numerical aperture greatly improves resolution to maximize the speed of acquisition per 3D image stacks. Crop the recording region of the EM CD's camera down to the immediate area of a cell pair. Furthermore, a large Z step between 0.45 and 0.75 micrometers may also help speed up the acquisition under these conditions.Images are continuously recorded at 20 to 60 minute intervals. Repeated imaging of the same sample spot can extend up to six hours with minimal photo bleaching when acquired. Each data segment is comprised of tens of thousands of images that may occupy a total of five to 20 gigabytes.To facilitate the transport and analysis of the large files, each file is broken down and exported as one gigabyte TIFF image File segments. To analyze the acquired images, use any of several image analysis software programs. Consult the accompanying written protocol for details of image analysis, including intensity bleach correction and automatic and manual tracking of gag puncta.Within 10 to 15 minutes of mixing, one can visualize HIV gag IGFP expressing jerk cat cells adhering to primary CD four positive T cells imaging. These conjugates one can assess the movements of gag puncta in infected cells as well as in target cells. After synapse, within 30 minutes to three hours, one may observe movement of gag IG FP puncta into target cells that are cell tracker labeled.We've just shown you how to visualize cell to cell transfer of HIV between T-cells. When doing these procedures, it's very important to remember to observe all biosafety level two plus or biosafety level three precautions when handling samples that have been exposed to HIV expressing cells. Also remember to use well maintained JCA cells and endotoxin free DNA to optimize transfect.So that's it. Thanks for watching and good luck with your experiments.

visualizing-cell-to-cell-transfer-hiv-using-fluorescent-clones-hiv

Research

JoVE Journal

Immunology and Infection

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Source: Lê-Bury, G., et al. Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1. J. Vis. Exp. (2016)This video demonstrates live confocal imaging of HIV-1-infected human macrophages to visualize and quantify phagosome migration using GFP fluorescence and bright field tracking, providing insights into how HIV alters intracellular trafficking during Fc receptor–mediated phagocytosis.

Live Imaging of Phagosome Migration in HIV-1-Infected Human Macrophages

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

Sample Preparation for In Situ Cryotomography of Mammalian Cells

Learn live confocal microscopy for visualizing HIV transfer across virological synapses between infected and uninfected T cells. This protocol enables real-time observation of viral transmission dynamics.

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

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