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. Preparation of Human Monocyte-derived Macrophages (hMDMs) by Density Gradient Centrifugation and Selection by Adhesion
<ol>
	<li>Start with fresh blood from healthy donors (9 ml). Dilute the entire volume of fresh blood with sterile 1x phosphate buffered saline (PBS) without Ca2+ and Mg2+ to obtain a final volume of 70 ml and gently add the diluted blood into two 50 ml conical tubes (35 ml per tube), on top of 15 ml of a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution already in each tube.</li>
	<li>Centrifuge both tubes of blood at 537 x g for 20 min at 20 &#176;C without brake. Then collect the peripheral blood mononuclear cells (PBMCs) contained in the cloudy cell ring at the interface and transfer them into a new 50 ml tube containing 15 ml of 1x PBS without Ca2+ and Mg2+.</li>
	<li>Centrifuge the cells at 218 x g for 5 min at 20 &#176;C and resuspend the pellet in 45 ml of 1x PBS without Ca2+ and Mg2+.</li>
	<li>Centrifuge the cells at 218 x g for 5 min at 20 &#176;C, resuspend the pellet in 10 ml of 1x PBS without Ca2+ and Mg2+, and count the cells by diluting to a final dilution of 1/200 in Trypan Blue.</li>
	<li>Centrifuge the cells at 218 x g for 5 min at 20 &#176;C and resuspend the pellet in RPMI (Roswell Park Memorial Institute) 1640 medium supplemented with 2 mM L-glutamine and 100 &#181;g/ml penicillin-streptomycin to have 7 x 106 PBMCs per well in 2 ml of medium in each well of a 6 well-plate.</li>
	<li>Incubate the plates at 37 &#176;C with 5% CO2 for 2 hr. After 2 hr the monocytes will have adhered to the plastic.</li>
	<li>To allow freshly-isolated monocytes to differentiate into hMDMs, aspirate the medium and replace it with 2 ml hMDM medium (RPMI 1640, 10% decomplemented Fetal Calf Serum (FCS), 2 mM L-glutamine and 1% penicillin-streptomycin) supplemented with recombinant human Macrophage Colony Stimulating Factor (rhM-CSF) at a final concentration of 10 ng/ml.</li>
	<li>Incubate the cells at 37 &#176;C with 5% CO2 for 11 days (Figure 1).</li>
	<li>At day 11 remove the medium and wash 2 times with 2 ml of cold hMDM medium per well. Next wash each well 2 times with 1 ml of cold 1x PBS.</li>
	<li>To detach fully differentiated hMDMs, wash each well 1 time with 1 ml of cold 1x PBS with 2 mM ethylenediaminetetraacetatic acid (EDTA) and incubate the cells in 2 ml of cold 1x PBS with 2 mM EDTA per well for 15-60 min at 4 &#176;C. 
	NOTE: Alternative methods to detach the cells exist (e.g., trypsin or trypsin-like activities) that might give good results 11.</li>
	<li>After detachment (completed by pipetting gently up and down in the well), collect the cells and put them in a 50 ml tube on ice containing 10 ml of cold hMDM medium.</li>
	<li>Centrifuge the cells at 218 x g for 5 min at 20 &#176;C, resuspend the pellet in 10 ml of cold hMDM medium, and count the cells by diluting 1/20 in Trypan Blue.</li>
	<li>Seed the cells at 1 x 106 hMDMs per 35 mm microscopy grade glass bottom dish and incubate the plates at 37 &#176;C with 5% CO2 for 1 day.</li>
</ol>
2. Production and Quantification of HIV-1 Viral Stocks
NOTE: NLR4.3 HIV-1 Gag-iGFP (Green Fluorescent Protein) carrying an R5-tropic envelope, gift from M. Schindler 10 is used to infect macrophages and to see infected cells in real time.
<ol>
	<li>Produce viral stocks by transfection of human embryonic kidney 293T cells (2 x 106 in 100 mm dish) with 6 &#181;g of the corresponding proviral DNA using a commercial transfection reagent.</li>
	<li>Quantify the infectivity of the virus stocks using the indicator cells HeLa TZM-bl (bearing the &#223;-galactosidase gene under the control of HIV-1 LTR) using serial dilutions of the viral stocks followed by a &#223;-galactosidase coloration of the cells and counting of blue cells 12.</li>
</ol>
3. Infection of hMDMs with HIV-1
<ol>
	<li>Add the virus at a multiplicity of infection (MOI) 0.02-0.03 in 1 ml of hMDM medium to hMDMs (cells plated in 1.13). To control wells add only 1 ml of hMDM medium and incubate the dishes at 37 &#176;C with 5% CO2 for 2 days (Figure 1).</li>
	<li>At day 2 wash the cells with hMDM medium 3 times and add 1 ml of fresh hMDM medium per dish. Incubate the cells at 37 &#176;C with 5% CO2 for 6 days (Figure 1).</li>
</ol>
4. Opsonization of Sheep Red Blood Cells
<ol>
	<li>For the preparation of 7 x 106 SRBCs per dish, wash the SRBCs two times in 100 &#181;l of solution containing 0.1% Bovine Serum Albumin (BSA) in 1x PBS with centrifugation at 600 x g for 4 min.</li>
	<li>Resuspend the washed SRBCs in 500 &#181;l 1x PBS/BSA 0.1% with rabbit IgG anti-SRBCs at a sub-agglutinating concentration per 5 &#181;l of SRBCs and incubate with rotation at RT for 30 min. 
	NOTE: To determine the sub-agglutinating concentration of anti-SRBCs IgG, prepare serial dilutions of IgG (stock concentration at 13.1 mg/ml) from 1/50 to 1/25,600 in 20 &#181;l in a 96-well plate. Add 2 x 106 SRBCs in 20 &#181;l in each well and put the plate in a dark room during several hr. The sub-agglutinating concentration is the dilution of the well just before the well with agglutination (IgG+SRBCs forming a network).</li>
	<li>After rotation, centrifuge the IgG-opsonized-SRBCs at 600 x g for 4 min and wash with 100 &#181;l of 1x PBS/BSA 0.1% with centrifugation at 600 x g for 4 min.</li>
	<li>Resuspend the IgG-opsonized-SRBCs in pre-warmed Phenol red-free RPMI medium supplemented with 2 mM L-glutamine and 1% penicillin-streptomycin (1 ml/dish).</li>
</ol>
5. Live Cell Video Microscopy Phagocytosis Assay
<ol>
	<li>Use a confocal imaging system such as a spinning disk system equipped with a heating chamber at 37&#176;C with CO2 passing through a bottle with water for humidification.</li>
	<li>Turn on the heating chamber prior to the experiment to have the microscope stage at 37 &#176;C before the beginning of the phagocytosis assay. Turn on the microscope and computer, and load the imaging software.</li>
	<li>Optimize the imaging settings such as scanning speed, magnification, resolution, etc. to have at least one cell per field and to image one frame every minute between 60 to 120 min. 
	NOTE: Here, the sample is imaged one frame every minute during 60 min with 63X lens.</li>
	<li>Place the imaging dish on the microscope stage. Adjust the focus and the location to find just one whole HIV-1 infected macrophage in the field. Use appropriate excitation/emission settings based on the used imaging system and probe. Include a bright field (BF) channel to observe phagosomes (Figure 1ii). Optimize the appearance of the different channel images by adjusting the percentage of transmitted light and exposure time. 
	NOTE: Here, NLR4.3 HIV-1 Gag-iGFP was excited using a 491 nm laser with 50 msec of time exposure with 20% of laser (Figure 1i).</li>
	<li>Remove the imaging dish and add 1 ml of SRBC suspension at 7 x 106 SRBCs/ml to the dish (Figure 1).</li>
	<li>Centrifuge at 500 x g for 2 min at RT to synchronize phagocytosis, record the time at the end of this centrifugation and return the dish to the stage.</li>
	<li>Optimize the focus and capture GFP and BF images in Z-stacks (throughout the thickness of the cell with a step-distance of 0.3 &#181;m &#8211; usually 20 planes) every minute for at least 1 hr. Save the time-lapse video in the native file-format of the used imaging system. 
	NOTE: Here, time-lapse movies were saved in the native format, *.stk files.</li>
</ol>
6. Analysis of the Time-lapse Movies
<ol>
	<li>For video editing, click on the drop down menu &#34;Apps&#34; and on the tab &#34;Review Multi Dimensional Data&#34; in the video editing software.
	<ol>
		<li>To open the file, click on &#34;Select Base File&#34; then on &#34;Select Directory&#34;. In the &#34;Data sets&#34; box, select the acquisition to analyze (in .nd format) and click on &#34;View&#34;. The data are represented in a table with time in the column and Z-plan in the line.</li>
		<li>To represent the infection in a Z-projection (Figure 2, left panels), select 491 nm wavelength in the &#34;Wavelengths&#34; box and click on the &#34;Z projection&#34; tab with &#34;all planes&#34;.</li>
		<li>To analyze a time sequence, select BF wavelength in the &#34;Wavelengths&#34; box and choose the optimal plane on the Z-axis to distinguish external SRBCs (Figure 2, red arrowhead), internal SRBCs (Figure 2, red circle) and the nucleus (Figure 2, blue circle).</li>
		<li>To save the video montages, click on &#34;Selection [X&#39;s]&#34; tab and then on &#34;Load Image(s)&#34;. Finally, save the loaded images in .tif format and open them next on ImageJ software.</li>
	</ol>
	</li>
	<li>Use the plugin &#34;Manual Tracking&#34; on the ImageJ software to measure the position of the nucleus and the different phagosomes observed in BF channel (Figure 3).
	<ol>
		<li>Download the &#34;Manual Tracking&#34; plugin on the ImageJ website. On ImageJ, open the plugin and the image sequence to be analyzed (Figure 3, step 1 and 2).</li>
		<li>Enter the settings such as &#34;Time Interval&#34; which represents amount of time between adjacent frames, and the &#34;x/y calibration&#34; which represents the distance per pixel (Figure 3, step 3). 
		NOTE: Here, save the image sequence with a time interval of 1 min between each frame and x/y calibration of 0.205 &#181;m because the 63X zoom and a camera with pixel size of 6.45 x 6.45 &#181;m2 are used.</li>
		<li>To start the tracking, click on &#34;Add track&#34; (Figure 3, step 4) and click on an SRBC center at the first time when it is internalized. The next frame appears automatically.</li>
		<li>Continue to click on the SRBC center in all frames to have different positions during the time (Figure 3, step 6 in red box).
		<ol>
			<li>Start to track the nucleus to have its position in all frames by clicking on its center. Next track the phagosomes (by clicking on its center) one by one at the time (frame) of their internalization, different in function of SRBCs. For convenience to see SRBC on BF channel, use the &#34;Brightness &#38; contrast&#34; window during the tracking (Figure 3, step 5).</li>
		</ol>
		</li>
		<li>Between each SRBC tracking, click on &#34;Add track&#34; (Figure 3, step 4) to have a new track. The number of the tracking will be changed in the second column, &#34;Track n&#176;&#34; of the results table (Figure 3, step 6).</li>
		<li>Save the data in a spreadsheet to continue to the next step of the analysis.</li>
	</ol>
	</li>
	<li>Use spreadsheet software to calculate the travelled distance of phagosomes containing SRBCs towards the nucleus and the velocity of the phagosomes during the first 5 min after internalization of SRBCs (Figure 4).
	<ol>
		<li>Open the spreadsheet table and a new spreadsheet file. Transfer into this new file the following parameters only, time, the x- and y- coordinate of the nucleus and all SRBCs (Figure 4A).</li>
		<li>To calculate the distance between SRBCs and the nucleus with only their coordinates, consider the nucleus and a SRBC in a XY-coordinate plane (Figure 4B). 
		NOTE: The distance between two points is the length of the path connecting them. In the plane, the distance between SRBC and the nucleus is given by the Pythagorean theorem.
		<ol>
			<li>If c (the distance between the nucleus and the SRBC) denotes the length of the hypotenuse and a and b denote the lengths of the other two sides, express Pythagoras&#39; theorem as the Pythagorean equation: 
			<img alt="Equation 1" src="/files/ftp_upload/54568/54568eq1.jpg" /> 
			NOTE: At the same time, on the orthonormal, the horizontal distance a is (xnucleus-xSRBC) and the vertical distance b is (ynucleus-ySRBC).</li>
			<li>Thus, calculate the distance between the nucleus and the SRBC (Figure 4A, purple box) by: 
			<img alt="Equation 2" src="/files/ftp_upload/54568/54568eq2.jpg" /> 
			NOTE: Multiply the obtained distance value (in pixels) by x/y calibration to have the distance in &#181;m. Here, the x/y calibration is 0.205 &#181;m.</li>
			<li>At each time, subtract the distance between the nucleus and the SRBC to the initial distance (Figure 4C).</li>
			<li>Plot the measurement against the time for the first 5 min. Apply a linear trend-line (Figure 5A) to the plot and determine the slope of the linear trend-line that represents the phagosome velocity towards the nucleus during the first 5 min after SRBC internalization.</li>
			<li>Collate the data to calculate the average and statistical error of the velocity values and plot them in an appropriate form using the appropriate software (Figure 5B).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cell+biology+of+phagocytosis%5BTitle%5D)+AND+%22Annu.+Rev.+Pathol%22%5BJournal%5D)">The cell biology of phagocytosis.</a> Annu. Rev. Pathol. 7, 61-98 (2012).</li>
<li>Niedergang, F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNiedergang%2C+F+%22Encyclopedia+of+Cell+Biology%22">Encyclopedia of Cell Biology</a>. 2, Academic Press. Waltham, MA. 751-757 (2016).</li>
<li>Blocker, A., Griffiths, G., Olivo, J. C., Hyman, A. A., Severin, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+microtubule+dynamics+in+phagosome+movement%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">A role for microtubule dynamics in phagosome movement.</a> J Cell Sci. 111 (Pt 3), 303-312 (1998).</li>
<li>Blocker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+requirements+for+bi-directional+movement+of+phagosomes+along+microtubules%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Molecular requirements for bi-directional movement of phagosomes along microtubules.</a> J Cell Biol. 137, 113-129 (1997).</li>
<li>Flannagan, R. S., Cosio, G., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+mechanisms+of+phagocytes+and+bacterial+evasion+strategies%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Antimicrobial mechanisms of phagocytes and bacterial evasion strategies.</a> Nat Rev Microbiol. 7, 355-366 (2009).</li>
<li>Feasey, N. A., Dougan, G., Kingsley, R. A., Heyderman, R. S., Gordon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invasive+non-typhoidal+salmonella+disease%3A+an+emerging+and+neglected+tropical+disease+in+Africa%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Invasive non-typhoidal salmonella disease: an emerging and neglected tropical disease in Africa.</a> Lancet. 379, 2489-2499 (2012).</li>
<li>Mazzolini, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+phagocytosis+in+HIV-1-infected+macrophages+relies+on+Nef-dependent+alteration+of+focal+delivery+of+recycling+compartments%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments.</a> Blood. 115, 4226-4236 (2010).</li>
<li>Dumas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+HIV-1+protein+Vpr+impairs+phagosome+maturation+by+controlling+microtubule-dependent+trafficking%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking.</a> J Cell Biol. 211, 359-372 (2015).</li>
<li>Greenberg, S., el Khoury, J., Kaplan, E., Silverstein, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+fluorescence+technique+to+distinguish+attached+from+ingested+erythrocytes+and+zymosan+particles+in+phagocytosing+macrophages%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">A fluorescence technique to distinguish attached from ingested erythrocytes and zymosan particles in phagocytosing macrophages.</a> J. Immunol. Methods. 139, 115-122 (1991).</li>
<li>Koppensteiner, H., Banning, C., Schneider, C., Hohenberg, H., Schindler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macrophage+internal+HIV-1+is+protected+from+neutralizing+antibodies%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Macrophage internal HIV-1 is protected from neutralizing antibodies.</a> J Virol. 86, 2826-2836 (2012).</li>
<li>Gartner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+macrophage+and+HIV%3A+basic+concepts+and+methodologies%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">The macrophage and HIV: basic concepts and methodologies.</a> Methods Mol Biol. 1087, 207-220 (2014).</li>
<li>Wei, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emergence+of+resistant+human+immunodeficiency+virus+type+1+in+patients+receiving+fusion+inhibitor+%28T-20%29+monotherapy%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</a> Antimicrob Agents Chemother. 46, 1896-1905 (2002).</li>
<li>Harrison, R. E., Bucci, C., Vieira, O. V., Schroer, T. A., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phagosomes+fuse+with+late+endosomes+and%2For+lysosomes+by+extension+of+membrane+protrusions+along+microtubules%3A+role+of+Rab7+and+RILP%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP.</a> Mol Cell Biol. 23, 6494-6506 (2003).</li>
<li>Toyohara, A., Inaba, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transport+of+phagosomes+in+mouse+peritoneal+macrophages%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Transport of phagosomes in mouse peritoneal macrophages.</a> J Cell Sci. 94 (Pt 1), 143-153 (1989).</li>
</ol>

The authors have nothing to disclose.

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 verified the expression of CD4 and CCR5. The infection rates are variable and range from 10 to 40%, and sometimes lower. This variable infection rate is why it can be difficult to find an HIV-1 infected fluorescent hMDM under the microscope. In addition, the time needed to find an HIV-1 infected cell immediately after the centrifugation step can also lead to a delay in starting the acquisition. Phagocytosis could be initiated without the synchronization of binding and uptake generated by centrifugation. However, this option was not selected for SRBCs that float before reaching the cells. It could be a good option with beads. To minimize the time needed to find infected cells, gridded glass bottom dishes could be used to identify HIV-1 infected hMDMs before proceeding to start the phagocytosis assay. The selected HIV-infected cells that have a &#34;reasonable&#34; number of SRBCs around them will then be quickly chosen to start the acquisitions. &#34;Reasonable SRBC amount&#34; means enough to have at least 10 SRBCs per cell and not too many to prevent the external SRBCs from masking the phagosomes.
In addition, it is important to optimize the opsonization conditions to ensure there will be efficient recognition and uptake by the cells. The FcR-mediated phagocytosis is constitutive and hMDMs are routinely very efficient in FcR-mediated uptake. In addition, for correct phagocytosis, the conditions have to be optimal with the heating chamber at 37 &#176;C and 5% CO2. It is necessary to check the gas and the temperature before starting the phagocytosis assay.
Finally the discrimination of external SRBCs from internal phagosomes and the identification of the nucleus in the BF channel are important for analysis on the ImageJ software. This is why the settings of this channel are important. Given that the focus can be lost during acquisition, we recommend to acquire stacks of images in the Z axis to be sure to have the optimal plane to analyze phagosome velocity. It is preferable to have one single cell in one field to analyze all internalized SRBCs for each cell. That is why the lens choice is important and here we have used 2 lenses as a function of the cell size. It was easier for us to track phagosomes with a 100X lens, but sometimes the cells were too large so we had to switch to a 63X lens. The choice of lens is also critical when thinking about image analysis using ImageJ and a spreadsheet table because both lenses implicate different x/y calibrations.
The first limitation of this technique is the number of samples that need to be acquired to have statistically significant results. We can register several movies per donor but usually not more than 2 cells per condition, per dish. The solutions to this limitation to increase the number of samples are to repeat experiments with more donors or to do multi-point XY imaging depending on the microscope. The inconvenience of the last method is the potential loss of focus during motorized XYZ scanning. Another limitation is that the manual quantification of velocity is long and tedious compared to an automated analysis. For full automated analysis, it is necessary to use fluorescently labelled particles, as reported in pioneering studies 4 with latex beads coupled to fish skin gelatin that were followed with a tracking algorithm on MatLab software. Fluorescently labeled opsonizing antibodies or beads could be an alternative, but they are subject to bleaching, which is a problem when recording phagocytosis for 1 hr. Other studies report the use of latex beads 13,14. We noticed however that internalization of latex beads is less easily detected than the uptake of SRBCs. Indeed the starting point of the kinetics of intracellular migration is important to know to detect early defects in phagosomal movement.
The main advantage of the method described here is its simplicity and the use of a free software. To perform the manual tracking using the ImageJ software, we have to use the position of the phagosome and the nucleus, thus, we need to use Pythagoras&#39; theorem to calculate the distance between the phagosome and the nucleus, and to have the velocity after a linear regression in spreadsheet software. This 3 steps analysis can be reduced to one step, the tracking, on ICY software with the &#34;Manual Tracking&#34; plugin. The output data are then directly the velocity of the particle to follow, and more particularly the relative velocity between 2 groups (such as phagosomes and nucleus). Finally, the calculation allows us to measure the relative velocity of one particle compared to another particle that can also be moving.
The potential future application of this living time-lapse analysis can be for particles visible in bright field, like bacteria, fungi, or cell debris. Different treatments or infection conditions sufficient to modify phagosomal maturation and the activation properties of macrophages would benefit from such a refined analysis of phagosomal movement towards the cell center.

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.

<table><tbody><tr><td>Falcon 100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td>For viral production</td></tr><tr><td>Glass Bottom Dishes 35 mm uncoated 1.5</td><td>MatTek corporation</td><td>P35G-1.5-14-C Case</td><td>For acquisition</td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Thermo Fischer Scientific</td><td>Corning. Inc. 353934</td><td>For human monocyte-derived macrophages</td></tr><tr><td>Ficoll-Plaque PLUS</td><td>Dominique Dutscher</td><td>17-1440-03</td><td>a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>RT</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) 1x, liquid (High Glucose)</td><td>GIBCO, Molecular probes</td><td>31966-021</td><td>Conserved at 4 &amp;deg;C ; for HEK cells culture</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td>Conserved at 4 &amp;deg;C; for hMDMs culture</td></tr><tr><td>F&amp;oelig;tal Calf Serum (FCS)&amp;nbsp;</td><td>Eurobio</td><td>CVFSVF0001</td><td>Conserved at -20 &amp;deg;C ; decomplemented</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Thermo Fischer Scientific</td><td>15140-122</td><td>Conserved at -20 &amp;deg;C ; for hMDMs culture</td></tr><tr><td>RPMI 1640 medium, no phenol red (10x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use ; for phagocytosis assay</td></tr><tr><td>FuGENE6 Transfection Reagent</td><td>Promega</td><td>E2692</td><td>Conserved at 4 &amp;deg;C ; for viral production</td></tr><tr><td>Sheep red blood cells (SRBCs)</td><td>Eurobio</td><td>DSGMTN00-0Q</td><td>Conserved in Alsever buffer at 4 &amp;deg;C before use</td></tr><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td>Conserved at 4 &amp;deg;C</td></tr><tr><td>Bovine Serum Albumin heat shock fraction, pH 7, &amp;ge;98%&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td>Conserved at -20 &amp;deg;C</td></tr><tr><td>Inverted microscope DMI600</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal Spinning Disk Unit&amp;nbsp; CSU-X1M1</td><td>Yokogawa</td><td></td><td></td></tr><tr><td>491 nm 50 mW laser</td><td>COBOLT CALYPSO</td><td></td><td></td></tr><tr><td>HCX PL APO CS Objectif</td><td>Leica</td><td></td><td>Objective lens ; Magnification 100X ; Numerical aperture 1.40 ; Immersion oil</td></tr><tr><td>CoolSnap HQ2 (FireWire) Camera</td><td>Photometrics</td><td></td><td>Pixel size 6.45 &amp;micro;m x 6.45 &amp;micro;m ; Definition 1,392 x 1,040 ; Encoding the image in 14 Bit</td></tr><tr><td>Metamorph 7.7.5 software</td><td>Molecular Devices</td><td></td><td>For the control of the microscope</td></tr><tr><td>GraphPad Prism software</td><td></td><td></td><td>For the statistics analysis</td></tr></tbody></table>

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 the erythrocytes, because this could induce their recognition and uptake as debris instead of by FcR-mediated phagocytosis. Optimal opsonization will ensure efficient phagocytosis.
Phagosome movement in living HIV-infected macrophages is analyzed in real-time with the BF channel on a spinning disk confocal microscope in the BSL-3 laboratory. The settings of the BF channel are critical to see the nucleus (Figure 2, blue circle) and for discriminating the external (Figure 2, red arrowheads) from the internalized SRBCs (Figure 2, red circles). The BF microscopy is considered as the simplest optical microscopy illumination technique sufficient to see the phagosomes, which are less refringent than the external SRBCs.
The first step after acquisition of image sequences is manual tracking on the ImageJ software (Figure 3). This point can be particularly long and tedious, because it is preferable to track each phagosome, one by one for all image sequences and it is considered that experiments need to be repeated with at least 3 donors per condition to reach a large enough sample size for statistical analysis (at least 30 phagosomes with 3 different donors).
The second step is the analysis in spreadsheet software to calculate the phagosome velocity during the first 5 min after internalization for each internalized SRBCs (Figure 4). For this, we plot for each phagosome the distance travelled during the first 5 min against the time. A representative example is shown in non-infected hMDMs on Figure 5A. Next we obtain the velocity of the phagosome, which is the slope of the linear regression curve. A velocity of 0.5384 &#181;m/min was found for this phagosome.
Of note, it is possible to analyze the phagosome velocity during the first few minutes after internalization, as described here or in Dumas et al. 8, or later by analyzing the velocity over longer timescales. In our study, we observed that the phagosome velocity during the first 5 min after internalization in HIV-infected hMDMs is lower compared to non-infected hMDMs (Figure 5B).
<img alt="Figure 1" src="/files/ftp_upload/54568/54568fig1.jpg" /> 
Figure 1. Preparation of control and HIV-infected hMDMs for phagocytosis imaging. Monocytes are purified by adhesion (1) and differentiated into macrophages for 11 days with M-CSF (2). Next, hMDMs are infected with NLR4.3 HIV-1Gag-iGFP i) at an MOI between 0.02 and 0.03 for 8 days with washing on day 2 (3). (4) IgG-opsonized SRBCs are added onto hMDMs for phagocytosis during 1 hr. The phagosomes containing SRBCs are detectable on BF images (red circle, ii). Images are from the Servier images database. Bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54568/54568fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54568/54568fig2.jpg" /> 
Figure 2. Real-time acquisition during phagocytosis by representative control and HIV-infected hMDMs. hMDMs are prepared and infected as described in Figure 1. The NLR4.3 HIV-1Gag-iGFP infected cells are detected with the 491 nm laser. Z-stacks of spinning disk confocal images are acquired and analyzed using both the microscope acquisition software and ImageJ (left panels). Galleries of BF images represent a non-infected (top panels corresponding to Movie 1) and a NLR4.3 HIV-1Gag-iGFP infected (bottom panels corresponding to Movie 2) cell during a 45 min acquisition (46 images with time indicated as hh:mm). The cell membrane (dotted black line), the nucleus (blue circle), the external SRBCs (some of them indicated with red arrowheads) and the internal SRBCs (some of them indicated with red circles) are detectable on BF images. Bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54568/54568fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54568/54568fig3.jpg" /> 
Figure 3. Instruction steps for image analysis with Manual Tracking plugin on ImageJ. After opening the &#34;Manual Tracking&#34; plugin (1) and loading the time-lapse video in BF channel (2), enter the parameters of the acquisition (3). Click on &#34;Add track&#34; (4) and start the measurement of the tracking by clicking on one phagosome (5) to obtain a new window with X and Y positions of the chosen phagosome (6, red box). To follow the phagosome on the time sequence for convenience, vary the brightness and contrast (5&#39;). <a href="https://www.jove.com/files/ftp_upload/54568/54568fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54568/54568fig4.jpg" /> 
Figure 4. Instruction steps for data analysis of phagosome migration velocity in spreadsheet software. (A) Assemble the X and Y position of the nucleus and each phagosome (red box) in a spreadsheet table. In another column (purple box), calculate the distance between the phagosome and the nucleus with Pythagoras&#39; theorem where c represents the length between the nucleus and the SRBC and a and b the lengths of the other two sides of a right-angled triangle (B). (C) Finally, calculate the distance travelled by the phagosomes at each time (green box) by subtracting the distance between the SRBC and the nucleus at a given time from the initial distance at time 0. <a href="https://www.jove.com/files/ftp_upload/54568/54568fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54568/54568fig5.jpg" /> 
Figure 5. Quantification of phagosome migration velocity in control and HIV-infected hMDMs. (A) For each SRBC and just after SRBC internalization, the travelled distance towards the nucleus is measured and plotted against the time. Its velocity during the first 5 min is calculated using linear regression in spreadsheet software. A representative experiment is shown (the internal SRBC of Figure 3-4). (B) All velocities during the first 5 min are measured for 66 phagosomes in non-infected (5 donors) and 60 phagosomes in HIV-infected cells (4 donors). Data represent the mean &#177; SEM (Unpaired Student t test with Welch&#39;s correction; **, P &#60;0,01). <a href="https://www.jove.com/files/ftp_upload/54568/54568fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/54568/54568movie1.jpg" /> 
<a href="https://www.jove.com/files/ftp_upload/54568/Jove_movie1.mov" target="_blank">Movie 1: Phagosome movement in a representative non-infected macrophage. (Right click to download).</a> The movement of opsonized red blood cells was detected in BF channel during 45 min of phagocytosis with one image per minute (46 images with time indicated as hh:mm). Bar = 10 &#181;m.
<img alt="Movie 2" src="/files/ftp_upload/54568/54568movie2.jpg" /> 
<a href="https://www.jove.com/files/ftp_upload/54568/Jove_movie2.mov" target="_blank">Movie 2: Phagosome movement in a representative HIV-infected macrophage. (Right click to download).</a> HIV-1 (NLR4.3 HIV-1 Gag iGFP)-infected macrophages were identified after illumination with the 491 laser. The movement of opsonized red blood cells was detected in BF channel during 45 min of phagocytosis with one image per minute (46 images with time indicated as hh:mm). Bar = 10 &#181;m.

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 ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.1K Views.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. 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. 

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

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 ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

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

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