We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

The protocol has to be carried out in strict accordance with national and international legislations and local regulations. Blood from healthy donors that gave their consent to donate blood for research purposes has been obtained from Blood Transfusion Centers with which the Institutions have signed agreement. Special protections must be taken when using human blood. Experiments with HIV-1 must be performed in a biosafety level 3 or 2 (BSL-3 or 2) laboratory according to local legislation.
1. Pr...

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This technique has several critical steps. Firstly, the preparation of hMDMs and their infection by HIV-1 is critical because the percentage of infection is donor dependent. Of note, we have decided to use macrophages that are not polarized in vitro prior to infection, because the status of macrophages potentially encountered by the virus in vivo has not been well characterized so far. We checked the expression of several surface markers, indicating that the macrophages are neither M1 nor M2, and verifi...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon 100mm TC-Treated Cell Culture Dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:111px;">353003</td>
			<td style="width:245px;">For viral production</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Glass Bottom Dishes 35 mm uncoated 1.5</td>
			<td style="width:124px;">MatTek corporation</td>
			<td style="width:111px;">P35G-1.5-14-C Case</td>
			<td style="width:245px;">For acquisition</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon Tissue Culture Plates 6-well</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">Corning. Inc. 353934</td>
			<td style="width:245px;">For human monocyte-derived macrophages</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Ficoll-Plaque PLUS</td>
			<td style="width:124px;">Dominique Dutscher</td>
			<td style="width:111px;">17-1440-03</td>
			<td style="width:245px;">a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">DPBS, no calcium, no magnesium</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">14190-094</td>
			<td style="width:245px;">Room temperature</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) 1X, liquid (High Glucose)</td>
			<td style="width:124px;">GIBCO, Molecular probes</td>
			<td style="width:111px;">31966-021</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for HEK cells culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">61870-010</td>
			<td style="width:245px;">Conserved at 4&#176;C; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">F&#339;tal Calf Serum (FCS)&#160;</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">CVFSVF0001</td>
			<td style="width:245px;">Conserved at -20&#176;C ; decomplemented</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">15140-122</td>
			<td style="width:245px;">Conserved at -20&#176;C ; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">11835-105</td>
			<td style="width:245px;">Warm in 37&#176;C water bath before use ; for phagocytosis assay</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">FuGENE6 Transfection Reagent</td>
			<td style="width:124px;">Promega</td>
			<td style="width:111px;">E2692</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Sheep red blood cells (SRBCs)</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">DSGMTN00-0Q</td>
			<td style="width:245px;">Conserved in Alsever buffer at 4&#176;C before use</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Anti-sheep red blood cells IgG</td>
			<td style="width:124px;">MP Biomedicals</td>
			<td style="width:111px;">55806</td>
			<td style="width:245px;">Conserved at 4&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:111px;">A7906</td>
			<td style="width:245px;">Conserved at -20&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Inverted microscope DMI600</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Confocal Spinning Disk Unit&#160; CSU-X1M1</td>
			<td style="width:124px;">Yokogawa</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">491 nm 50mW laser</td>
			<td style="width:124px;">COBOLT CALYPSO</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">HCX PL APO CS Objectif</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Objective lens ; Magnification 100x ; Numerical aperture 1.40 ; Immersion oil</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">CoolSnap HQ2 (FireWire) Camera</td>
			<td style="width:124px;">Photometrics</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Pixel size 6.45 x 6.45 &#181;m ; Definition 1392 x 1040 ; Encoding the image in 14 Bit</td>
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		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Metamorph 7.7.5 software</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the control of the microscope</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">GraphPad Prism software</td>
			<td style="width:124px;"></td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the statistics analysis</td>
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phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

FcR-mediated phagocytosis by HIV-1-infected and non-infected hMDMs is described here using IgG-opsonized SRBCs as model targets (Figure 1). The critical steps of this protocol are the preparation of hMDMs and the infection with HIV-1. Indeed, the yield and quality of differentiated macrophages varies among donors, as well as the infection rate with efficiencies in the range of 10-40%. In addition, the preparation of IgG-opsonized-SRBCs is also important to avoid damaging ...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

The overall goal of this analysis is to quantify phagosome velocity in HIV-1 infected macrophages using a simple manual tracking method. The advantage of this protocol is to provide an easy method to calculate the relative movements of groups of objects within a cell that can be either moving or deformed. This method provides insight into the movement of phagosomes in GFP-positive infected macrophages but it could be used for other intercellular compartments in fluorescently tagged cells.After infecting human monocyte-derived macrophages and opsonizing sheep-bred blood cells, according to the text protocol, set up a phagocytosis assay using a confocal imaging system, such as a spinning disc microscope, equipped with a heating chamber at 37 degrees Celsius, with carbon dioxide. Prior to the experiment, turn on the heating chamber to heat the microscope stage to 37 degrees Celsius. Then turn on the microscope and computer, and load the imaging software.Optimize the imaging settings, such as scanning speed, magnification, resolution, et cetera, to have at least one cell per field, and to image one frame every minute between 60 to 120 minutes. Place the imaging dish on the microscope stage. Adjust the focus and the location to find only one whole HIV-1 infected macrophage in the field.Use appropriate excitation and emission settings based on the used imagine system and probe. Include a bright field channel. Optimize its appearance by adjusting the exposure time.Next, remove the imaging dish and add one milliliter of sheep-bred blood cell, or SRBC suspension, at seven times tenth of the sixth SRBCs per milliliter, to the dish. Centrifuge the dish at 500 times g for two minutes at room temperature to synchronize phagocytosis, then record the time at the end of the centrifugation, and return the dish to the stage. Optimize the focus and set the top and bottom of the stack with a step distance of 0.3 micrometers.Start acquisition of the Z stacks of GFP and BF images throughout the thickness of the cell, and save the time-lapse video in the native file format of the imaging system. To carry out video editing, in the video editing software, click on the drop-down menu Apps and on the tab Review Multidimensional Data. To open the file, click on Select Base File, then on Select Directory.In the Data Sets box, select the acquisition to analyze and click on View. To represent the infection in a Z projection, in the Wavelengths box, select the 491 nanometer wavelength and click on the Z projection tab with all planes. Next, to analyze a time sequence, in the Wavelengths box select BF wavelength and choose the optimal plane on the Z axis to distinguish external SRBCs, internal SRBCs, and the nucleus.Save the video montages by clicking on the Selection Xs tab and then on Load Images. Finally, save the loaded images in tif format. After downloading the Image J manual tracking plugin, open the plugin in Image J, then open the image sequence to be analyzed.Enter the settings, such as time interval, which represents the amount of time between adjacent frames, and the XY calibration, which represents the distance per pixel. To start the tracking, click on Add Track, and click on an SRBC center at the first time when it is internalized. The next frame appears automatically.For convenience, to see SRBCs on the BF channel, use the brightness and contrast window during the tracking. Continue to click on the SRBC center in all frames to have different positions during the time. Between each SRBC tracking, click End Track and then Add Track to begin a new track.The tracking number will change in the second column of the results table. Start to track the nucleus to have its position in all frames by clicking on its center as performed previously. Save the data in a spreadsheet, then open it in spreadsheet software, and create a new spreadsheet file.Transfer into this new file the time, and the X and Y coordinates of the SRBC and the nucleus. Finally, use the spreadsheet software to calculate the traveled distance of phagosomes containing SRBCs towards the nucleus, and the velocity of the phagosomes during the first fives minutes after internalization of SRBCs according to the text protocol. Analysis of phagosome movement in living HIV-infected macrophages in real time using spinning disc confocal microscopy, is shown here.The settings for the bright field channel are critical to see the nucleus and for discriminating the external from the internalized SRBCs. This graph plots the distance traveled for a phagosome in non-infected HMDMs in the first five minutes after internalization of an SRBC versus time. The velocity of the phagosome is the slope of the linear regression curve shown here.Finally, in this study, it was observed that the phagosome velocity during the first five minutes after internalization in HIV-infected HMDMs is lower compared to non-infected HMDMs. These methods demonstrated that phagosome movement to the cell center, and therefore phagosome maturation into phagolysosomes, was impaired in HIV-infected macrophages. After its acquisition, a cell can be further processed for correlative electron microscopy or the whole-cell population can be fixed for classical immunofluorescence microscopy and statistical analysis.Phagocytosis is very sensitive to temperature and therefore the temperature within the heating chamber and the culture dish medium should be maintained at a constant 37 degrees Celsius throughout the procedure. Don't forget that working with pathogens can be hazardous and precautions such as working in a bio-safety room, and wearing personal protection equipment should be taken according to local legislation.

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Immunology and Infection

8.1K vistas.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. The overall goal of this analysis is to quantify phagosome velocity in HIV-1 infected macrophages using a simple manual tracking method. The advantage of this protocol is to provide an easy method to calculate the relative movements of groups of objects within a cell that can be either moving or deformed. This method provides insight into the movement of phagosomes in GFP-positive infected macrophages but it could be used for other intercellular compartments in fluorescently tagged cells....