We thank Gon&#231;alo Moniz Institute, Fiocruz Bahia, Brazil and the department of microscopy for assistance. This work was supported by INOVA-FIOCRUZ number 79700287000, P.S.T.V. holds a grant for productivity in research from CNPq (305235/2019-2). Plasmids were kindly provided by Mauricio Terebiznik, University of Toronto, CA. The authors would like to thank Andris K. Walter for English language revision and manuscript copyediting assistance.

Cells were obtained from healthy donors following the approval of procedures by the National Research Ethics Committees (ID: 94648218.8.0000.0040).
1. Cell cultures
<ol>
	<li>Human monocyte-derived macrophages 
	NOTE: To obtain human monocyte-derived macrophages for in vitro differentiation into macrophages, collect blood from healthy donors and purify peripheral blood mononuclear cells (PBMC) as described by D. English and B. R. Andersen21.
	<ol>
		<li>After collecting peripheral blood (50 mL), pour it into a heparinized tube and then dilute the blood 1:1 in a phosphate buffer solution (PBS) at room temperature. Gently place diluted heparinized blood on top of previously distributed density gradient medium.</li>
		<li>Centrifuge the tubes at 252 &#215; g for 30 min at 24 &#176;C to avoid hemolysis. 
		NOTE: Set centrifuge break-off to avoid mixing of gradient layers. After centrifugation, discontinuous gradient layers are formed from the bottom to the top: erythrocytes, density gradient medium, PBMC ring and plasma.</li>
		<li>Transfer the PBMC ring, located between the density gradient medium and plasma layers, to a new tube and fill with PBS to wash out excess density gradient medium.</li>
		<li>Wash cells once and centrifuge at 190 &#215; g for 10 min at 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend pellet in 1 mL of complete RPMI medium.</li>
		<li>Count the cells and plate 2 &#215; 106 cells in 500 mL of Roswell Park Memorial Institute (RPMI) supplemented with 25 mM N-[2-hydroxyethyl] piperazine-N&#8242;-[2-ethane sulfonic acid] (HEPES), 2 g/L sodium bicarbonate, 2 mM glutamine, 20 g/mL ciprofloxacin and 10% inactivated Fetal Bovine Serum (FBS) (complete RPMI medium) for 7 days at 37 &#176;C under 5% CO2 in a 24-well plate to allow monocytes to differentiate into macrophages by adhesion.</li>
	</ol>
	</li>
	<li>THP-1 cultures
	<ol>
		<li>Grow THP-1 cell line at a concentration of 2 &#215; 105 cells in 10 mL of complete RPMI medium in 75 cm2 culture flask.</li>
		<li>Maintain cell cultures in an incubator at 37 &#176;C under 5% CO2 for 7 days.</li>
		<li>Centrifuge cells at 720 &#215; g for 10 min at 4 &#176;C and resuspend the pellet in complete RPMI medium.</li>
		<li>Count cells in a Neubauer chamber.</li>
		<li>Plate cells on 13 mm glass coverslips at a concentration of 2 &#215; 105 cells per well in 500 &#956;L of complete RPMI medium containing 100 nM phorbol myristate acetate (PMA) at 37 &#176;C under 5% CO2 to allow differentiation of THP1 cells into macrophages.</li>
		<li>After three days, wash twice cells with 0.9% NaCl solution to remove medium containing PMA.</li>
		<li>Incubate differentiated THP-1 cells in PMA-free complete RPMI medium at 37 &#176;C under 5% CO2 for an additional 2 days before starting experimentation.</li>
	</ol>
	</li>
</ol>
2. Parasite cultures and CellTracker Red staining
NOTE: To visualize parasites through fluorescence microscopy, perform staining using CellTracker Red fluorescent dye (CMTPX). Alternatively, other markers, including carboxyfluorescein can be used in accordance with manufacturer instructions or promastigotes constitutively expressing GFP, RFP, or other fluorescent reporter genes. Parasites used to infect cells are those at stationary phase of growth obtained from a promastigote axenic culture of no more than 7 passages.
<ol>
	<li>Grow Leishmania spp. promastigotes at 1 x 105 parasites per 1,000 &#956;L of medium in a cell culture flask containing 5 mL of Schneider&#39;s medium supplemented with 50 &#956;g/mL gentamicin and 10% FBS.</li>
	<li>After incubating parasite axenic cultures in a biochemical oxygen demand (B.O.D.) at 24 &#176;C, perform daily counting in a Neubauer chamber. Check for parasite form (thin, elongated) and mobility for 5 days. Parasites are considered in stationary phase of growth when two consecutive counts with 8 hours of interval display similar amounts.</li>
	<li>Upon reaching the stationary phase of growth, incubate the parasites in 4 mL of 0.9% NaCl solution with 1 &#956;M CMTPX for 15 min at 37 &#176;C under 5% CO2 avoiding contact with light.</li>
	<li>Add FBS at a 1:1 proportion and incubate parasite suspension for an additional 1 min.</li>
	<li>Wash parasites thrice with PBS, followed by centrifugation at 1,781 &#215; g for 10 min.</li>
	<li>Ressuspend parasite pellet in 1,000 &#956;L of RPMI complete medium.</li>
	<li>Count parasites in a Neubauer chamber.</li>
</ol>
3. Assessment of Leishmania binding to macrophages
<ol>
	<li>Seed 2 &#215; 105 THP-1 cells or human monocyte-derived macrophages in 500 &#956;L of complete RPMI medium per well on a 24-well plate with 13 mm glass coverslips.</li>
	<li>Cultivate cells at 37 &#176;C under 5% CO2 for 24 h.</li>
	<li>Wash the cells twice with 0.9% NaCl solution and incubate in complete RPMI medium at 4&#160;&#176;C for 10 min.</li>
	<li>Add stationary phase promastigotes as described by A. L. Petersen22 at a 10:1 ratio to well plates, and then centrifuge at 720 &#215; g for 5 min under 4 &#176;C.</li>
	<li>Incubate at 4 &#176;C for 5 min.</li>
	<li>Wash the cells twice with 0.9% NaCl solution to remove any non-internalized promastigotes.</li>
	<li>Fix the cells in 4% paraformaldehyde for 15 min at room temperature.</li>
	<li>Incubate the coverslips with 15 mM NH4Cl for 15 min at room temperature.</li>
	<li>Wash thrice with PBS 0.15% bovine serum albumin (BSA). Incubate with blocking solution (3% BSA in PBS) for 1 h at room temperature.</li>
	<li>Wash thrice with PBS and then permeabilize with 0.15% PBS-Saponin for 15 min at room temperature.</li>
	<li>Add phalloidin (diluted 1:1,200) for 1 h at room temperature and protect from light.</li>
	<li>Mount coverslips using mounting media.</li>
	<li>Acquire images via a confocal fluorescence microscope using a 63&#215;/1.4 objective.</li>
</ol>
4. Assessment of Leishmania phagocytosis by macrophages
<ol>
	<li>Seed 2 &#215; 105 THP-1 cells or human monocyte-derived macrophages in 500 &#956;L of complete RPMI medium per well on a 24-well plate with 13 mm glass coverslips.</li>
	<li>Cultivate cells for 24 h at 37 &#176;C under 5% CO2.</li>
	<li>Wash cells twice in 0.9% NaCl solution and incubate in complete RPMI medium in 24-well plate at 4 &#176;C for 10 min.</li>
	<li>Add stationary phase Leishmania spp. as described by A. L. Petersen22 at a 10:1 (parasite:host cell) ratio, and then centrifuge at 720 &#215; g for 10 min under 4 &#176;C.</li>
	<li>Incubate cells at 4 &#176;C for 5 min.</li>
	<li>Wash the cells twice with 0.9% NaCl solution to remove any non-internalized promastigotes.</li>
	<li>Incubate the cells in supplemented RPMI medium at 37 &#176;C for 1 h.</li>
	<li>Fix the cells with 4% paraformaldehyde for 15 min.</li>
	<li>Mount coverslips using preferred mounting media.</li>
	<li>Count no less than 400 cells in random fields under a fluorescence microscope using a 100&#215;/1.4 objective.</li>
</ol>
5. Evaluation of Leishmania -induced vacuole maturation
NOTE: THP-1 cell transfection should be performed as described by M. B. Maess, B. Wittig and S. Lorkowski 23. Here we summarize this protocol, with minimal modifications. Nucleofection is a specific transfection method that requires a nucleofector. As an alternative method, cells can be transfected using lipofectamine24 and lentivirus transduction25.
<ol>
	<li>To investigate the biogenesis of Leishmania-induced PV, transfect THP1 cells with PLC26,27, Akt26,27, Rab 528,29,30 or Rab 728,29,31 plasmids. 
	&#8203;NOTE: This methodology can be used to transfect THP-1 cells with other genes than those listed above.</li>
	<li>Seed THP-1 cells at 1.5 x 107 in 75 cm&#178; tissue culture flasks containing 10 mL complete RPMI medium supplemented with 100 ng/mL PMA and 50 &#181;M 2-mercaptoethanol for 48 h.</li>
	<li>Wash cells once in 0.9% NaCl solution.</li>
	<li>Detach cells using a non-enzymatic cell dissociation solution and centrifuge (250 &#215; g) for 5 min at room temperature.</li>
	<li>Resuspend THP-1 cells in 1 mL of RPMI medium and perform counts in a Neubauer chamber.</li>
	<li>Centrifuge THP-1 cells again at 250 &#215; g for 10 min at room temperature. Discard the supernatant.</li>
	<li>Resuspend 2 &#215; 106 cells in 100 &#181;L of Nucleofector solution and incubate with 0.5 &#181;g of the plasmid coding for the protein of interest, tagged with a fluorescent protein.</li>
	<li>Transfer the suspension containing THP-1 cells and nucleic acid to the Nucleofector cuvette.</li>
	<li>Transfect THP1 cells using Nucleofector Program Y-001.</li>
	<li>Recover the transfected cells (2x106) and seed in 500 &#956;L RPMI medium on 24-well plates with 13 mm glass coverslips (4 wells/transfection).</li>
	<li>Incubate THP-1 cells in complete RPMI medium at 37 &#176;C for 0.5, 2, 4, 6, 12 and 24 h.</li>
	<li>Repeat steps 3.13 and 3.13.</li>
</ol>
6. Evaluation of the recruitment of LC3 to Leishmania spp. PVs
NOTE: The autophagic membrane marker LC3 can be used to investigate whether phagosomes present autophagic features. LC3 recruitment to Leishmania-induced PVs can be assessed during infection by immunolabelling cells with the anti-LC3 antibody, as previously described by C. Matte32 and B. R. S. Dias33.
<ol>
	<li>Seed 2 &#215; 105 THP-1 cells or human monocyte-derived macrophages in 500 &#956;L complete RPMI medium on a 24-well plate with 13 mm glass coverslips.</li>
	<li>Cultivate cells for 24 h at 37 &#176;C under 5% CO2.</li>
	<li>Wash cells twice in 0.9% NaCl solution and incubate in complete RPMI medium.</li>
	<li>Add stationary phase Leishmania spp. promastigotes as described by A. L. Petersen22 at a 10:1 (parasite: host cells) ratio and centrifuge cells at 720 &#215; g for 5 min under 4 &#176;C.</li>
	<li>Incubate at 37 &#176;C for 30 min or 4 h. Then wash twice and fix the cells to evaluate the LC3 recruitment to Leishmania-induced PV membranes at the early stages of infection.
	<ol>
		<li>Alternatively, to assess LC3 recruitment to PV membranes at later stages of infection, wash twice another macrophage group at 4 h of infection to remove any non-internalized promastigotes. Incubate infected cells in complete RPMI medium for an additional 12 h and 24 h, to finally wash twice and fix. 
		NOTE: Fixed cells can be kept in PBS or 0.9% NaCl solution at 4 &#176;C until labeling.</li>
	</ol>
	</li>
	<li>Simultaneously block and permeabilize the fixed cells in 0.1% Triton X-100, 1% BSA, 20% normal goat serum, 6% non-fat dry milk, and 50% FBS for 20 min at room temperature.</li>
	<li>Incubate the cells with anti-LC3 antibody (1: 200) diluted in PBS for 2 h at room temperature. 
	NOTE: As a negative control of the immunostaining, a group of cells should be incubated with immunoglobulin G (IgG) from the animal of primary antibody origin in a concentration equivalent to that used for the primary antibody.</li>
	<li>Wash the cells thrice with 0.9% NaCl solution at room temperature.</li>
	<li>Incubate the cells with AlexaFluor 488-conjugated goat anti-rabbit IgG (1:500) or the preferred fluorescent-dye conjugated secondary antibodies for 1 h at room temperature.</li>
	<li>Wash the cells thrice with 0.9% NaCl solution at room temperature.</li>
	<li>Mount coverslips using preferred mounting media.</li>
	<li>Acquire images via confocal fluorescence microscope using a 63x/1.4 objective.</li>
</ol>
7. Confocal microscopy acquisition and Fiji quantification
NOTE: Acquiring immunofluorescence images should be performed using a confocal laser scanning microscope. To reach a better resolution, use an oil-immersion 63x objective lens.
<ol>
	<li>Leave the 13 mm glass coverslips at room temperature and protect them from the light at least 30 min before the acquisition.</li>
	<li>Clean the coverslips with an absorbent tissue.</li>
	<li>Add a drop of immersion oil to the objective and add the slide.</li>
	<li>Move the objective up until the oil touches the slide.</li>
	<li>Observe and adjust the focus on the microscope and choose the option 63x objective with oil.</li>
	<li>Open the Leica program and adjust the lasers in the 488, 552, and 405 wavelengths.</li>
	<li>Select the image resolution 1,024 x 1,024.</li>
	<li>Click on the Live button, set the Z stack, and press the Begin option. Then, do it again and press the End button. We recommend 20 &#956;m for slice thickness to get confocal images with good resolutions.</li>
	<li>Wait for the image acquisition, and then select the option &#34;Maximum Projection&#34; in the Leica tools.</li>
	<li>Save the experiment.</li>
	<li>Export the lif or tiff format images to a computer and open the FIJI program.</li>
	<li>Open the experiment and set the view stack with the hyper stack. Then select open files individually and stitch tiles.</li>
	<li>Select the free hands tool in the Fiji toolbar and trace the cell carefully by hand.</li>
	<li>Press the Analyze button and measure to visualize the fluorescence intensity.</li>
	<li>Repeat this process to each cell per group.</li>
	<li>Save the measurements and export them to a spreadsheet editor.</li>
	<li>Add this data to a statistical analysis program and do the statistical analysis.</li>
</ol>
8. Statistical analysis
NOTE: For data analysis and graphics, use a statistical analysis program.
<ol>
	<li>Open the program.</li>
	<li>Insert the obtained data and test the normality parameters.</li>
	<li>For data with normal distribution, use the Student t-test and for nonparametric tests, Mann-Whitney test.</li>
	<li>Consider data with a statistically significant difference when the p-value is less than 0.05.</li>
	<li>Prepare graphics representing the data, with central tendency measures (mean or median) and variation measures.</li>
</ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autophagic+pathway+is+actively+modulated+by+phase+II+Coxiella+burnetii+to+efficiently+replicate+in+the+host+cell.">The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell.</a> Cellular Microbiology. 9 (4), 891-909 (2007).</li><li>Vieira, O. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+Rab5+and+Rab7+recruitment+to+phagosomes+by+phosphatidylinositol+3-kinase.">Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase.</a> Molecular and Cellular Biology. 23 (7), 2501-2514 (2003).</li><li>Roberts, R. L., Barbieri, M. A., Ullrich, J., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+rab5+activation+in+endocytosis+and+phagocytosis.">Dynamics of rab5 activation in endocytosis and phagocytosis.</a> Journal of Leukocyte Biology. 68 (5), 627-632 (2000).</li><li>Vitelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+small+GTPase+Rab7+in+the+late+endocytic+pathway.">Role of the small GTPase Rab7 in the late endocytic pathway.</a> Journal of Biological Chemistry. 272 (7), 4391-4397 (1997).</li><li>Matte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+major+Promastigotes+Evade+LC3-Associated+Phagocytosis+through+the+Action+of+GP63.">Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.</a> PLoS Pathogens. 12 (6), 1005690 (2016).</li><li>Dias, B. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+Induction+Greatly+Enhances+Leishmania+major+Intracellular+Survival+Compared+to+Leishmania+amazonensis+in+CBA/j-Infected+Macrophages.">Autophagic Induction Greatly Enhances Leishmania major Intracellular Survival Compared to Leishmania amazonensis in CBA/j-Infected Macrophages.</a> Frontiers in Microbiology. 9, 1890 (2018).</li><li>Babcock, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+phagocytosis+by+confocal+microscopy.">Quantitation of phagocytosis by confocal microscopy.</a> Methods in Enzymology. 307, 319-328 (1999).</li><li>Sanderson, M. J., Smith, I., Parker, I., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Cold Spring Harbor Protocols. 2014 (10), 071795 (2014).</li><li>Lennartz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+and+phagocytosis:+the+role+of+phospholipid-derived+second+messengers+in+phagocytosis.">Phospholipases and phagocytosis: the role of phospholipid-derived second messengers in phagocytosis.</a> International Journal of Biochemistry &amp; Cell Biology. 31 (3-4), 415-430 (1999).</li><li>Rashidfarrokhi, A., Richina, V., Tafesse, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+Early+Stages+of+Phagocytosis.">Visualizing the Early Stages of Phagocytosis.</a> Journal of Visualized Experiments. (120), e54646 (2017).</li><li>Ramarao, N., Meyer, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+resists+phagocytosis+by+macrophages:+quantitative+assessment+by+confocal+microscopy+and+fluorescence-activated+cell+sorting.">Helicobacter pylori resists phagocytosis by macrophages: quantitative assessment by confocal microscopy and fluorescence-activated cell sorting.</a> Infection and Immunity. 69 (4), 2604-2611 (2001).</li><li>Bain, J., Gow, N. A., Erwig, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+into+host-fungal+pathogen+interactions+derived+from+live-cell+imaging.">Novel insights into host-fungal pathogen interactions derived from live-cell imaging.</a> Seminars in Immunopathology. 37 (2), 131-139 (2015).</li></ol>

The funders had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Leishmania-macrophage interaction is a complex process and involves several steps that can influence disease development5. To better understand the mechanisms involved in the interaction of unopsonized Leishmania and host cells, we have described a protocol that employs confocal fluorescence microscopy to assess phagocytosis from early to late stages of Leishmania infection. The use of fluorescence techniques involving two or more fluorophores to investigate cell biology...

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania, resulting in a broad spectrum of clinical manifestations in the vertebrate host, including cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis1.&#160;The World Health Organization (WHO) estimates that over one billion people are at risk, with more than one million new cases reported per year2.
Leishmania spp. are obligate intracellular protozoans that survive inside host cells, including monocytes, macrophages and dendritic cells3. Leishmania-macrophage interaction is a complex process that involves multiple host cell receptors and parasite ligands either through direct interaction or by opsonization involving complement receptors4,5. Classical surface receptors, such as CR1, CR3, mannose-fucose, fibronectin, toll-like and scavenger receptors, mediate parasite attachment to macrophages6,7,8. These receptors recognize molecules on the surface of Leishmania, including the 63 kDa glycoprotein (gp63) and glycolipid lipophosphoglycan (LPG)9. These are the most abundant molecules on the surface of promastigotes and play an essential role in the subversion of host immune response, favoring the establishment of parasite infection in mammalian cells10. After parasite surface ligands bind to macrophage receptors, F-actin accumulates on mammalian cell surfaces, surrounding parasites as they are phagocytosed. Subsequently, this leads to the formation of a parasite-induced compartment termed parasitophorous vacuole (PV), which presents phagolysosomal features11. Once inside these phagolysosomes, parasites undergo several alterations essential to survival and multiplication3.
The biogenesis of PVs is a highly regulated membrane trafficking process critical to the intracellular survival of this pathogen12. The formation of this compartment results from sequential fusion events between phagosomes and compartments of the host endocytic pathway. Classical cell biology studies have revealed that the maturation of PVs involves the acquisition of monomeric G protein Rab 5 and Rab 7 proteins, which are mainly associated with early and late endosome maturation, respectively13. In addition, these compartments acquire lysosome-associated membrane proteins 1 and 2 (LAMP 1, LAMP 2), the principal protein constituents of the lysosomal membrane and microtubule-associated protein 1A/1B-light chain 3 (LC3), an autophagosome marker14. Despite apparent similarities, the kinetics of PV formation15,16 and the morphology of these compartments vary depending on Leishmania species. For example, infection caused by L. mexicana or L. amazonensis induces the formation of large compartments containing a great number of parasites17. By contrast, other species, such as L. braziliensis and L. infantum, form smaller vacuoles that normally contain only one or two parasites in each vacuole18.
Despite this knowledge surrounding host cell-Leishmania interaction, the initial events triggered by contact between host receptors and parasite ligands have not been fully elucidated. These events are known to be determinants of the outcome of parasite infection and are dependent on parasite species, the type of host cell receptors recruited to recognize parasites and the activation of macrophage signaling pathways19,20. Therefore, it is essential to identify the molecules involved in the biogenesis of Leishmania-induced PVs and determine the role(s) played by these molecules in infection establishment and outcome. Here, we describe a method of monitoring early events occurring during the phagocytosis of Leishmania, including binding, internalization, phagosome formation and maturation. This work could aid in clarifying the participation of PLC, Akt, Rab5, Rab7 and LC3 in the formation of PVs induced by different Leishmania species. Importantly, this protocol can be used to investigate the participation of other proteins involved in PV maturation. Future studies will expand the knowledge surrounding mechanisms involved in Leishmania-host cell interaction and contribute to the design of novel chemotherapeutic strategies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488-conjugated goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>Tem varios no site</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-LC3 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB600-1384</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>CellStripper</td>
			<td>Corning</td>
			<td>25-056-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red (CMTPX) Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>Centr&#237;fuga</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ciprofloxacin</td>
			<td>Isofarma</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal fluorescence microscope (Leica SP8)</td>
			<td>Leica</td>
			<td>Leica SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope (Olympus Lx73)</td>
			<td>Olympus</td>
			<td>Olympus Lx73</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (N- 2-hydroxyethyl piperazine-N&#8217;-2-ethane-sulfonic acid)</td>
			<td>Gibco</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma</td>
			<td>NS02L</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector 2b Device</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleofector solution</td>
			<td>Lonza</td>
			<td>VPA-1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol myristate acetate (PMA)</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer solution (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade kit</td>
			<td>Life Technologies</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Thermo Fisher Scientific</td>
			<td>X</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect medium</td>
			<td>Sigma</td>
			<td>S0146</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This report aims to evaluate the early events occurring during the phagocytosis of L. braziliensis isolated from patients presenting L. braziliensis-LCL or L. braziliensis-DL form of CL. Using confocal microscopy, we investigated the main events associated with parasites&#39; phagocytosis: binding, internalization, and phagosome maturation. We first evaluated the L. braziliensis-LCL or L. braziliensis-DL binding and phagocytosis by human monocyte-derived macrophages. The data ...

Watch this Scientific Journal Video about Investigating the Phagocytosis of Leishmania using Confocal Microscopy at JoVE.com

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.

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 ...

investigating-the-phagocytosis-of-leishmania-using-confocal-microscopy

Research

JoVE Journal

Immunology and Infection

2.8K Views.  Gonçalo Moniz Institute. 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.

Video: Investigating the Phagocytosis of Leishmania using Confocal Microscopy

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

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 ...

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

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 ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...