This work was supported by the National Institute of Allergy and Infectious Disease Grant R01AI079253 (to B.H.S.), and by the National Cancer Institute Cancer Center Support Grant CA016056 to Roswell Park Cancer Institute.

All procedures performed on animals in this study were approved by the Animal Care and Use Committee at Roswell Park Cancer Institute and complied with all state, federal, and National Institutes of Health regulations.
1. In vivo Macrophage Labeling
Note: PHK26 is a lipophilic dye that will be taken up by phagocytic cells both in circulation and in the tissues when administered intravenously (i.v.). PKH26 was used for in vivo labeling to minimize the amount of post-harvest manipulation of the macrophage. PKH-26 has the advantage of stably accumulating in alveolar macrophages over prolonged periods. Waiting 10 days after administration allows for clearance of all labeled cells from circulation leaving only alveolar macrophage stably labeled with PKH268,9. PKH26 is a red fluorochrome that has excitation (551 nm) and emission (567 nm) characteristics compatible with rhodamine or phycoerythrin detection systems. Any labeling procedure can introduce artifact, including loss of viability. However, since we use the same approach for labeling of macrophages in different mouse genotypes (e.g., WT vs. NADPH oxidase-deficient), the effects of these artifacts would be uniform.
<ol>
	<li>Dilute stock solution of PKH26 (1 x 10-3 M in ethanol) 1:5 in diluent C provided by manufacturer.</li>
	<li>Load 100 &#181;l of diluted PKH26 into a 1/2 ml insulin syringe (29 G x 1/2&#8221;) for each mouse.</li>
	<li>Administer to mice by i.v. injection (either tail vein or retro orbital) at least 10 days prior to harvest.</li>
</ol>
2. Harvesting Alveolar Macrophages by Bronchoalveolar Lavage (BAL)
The number of mice to use for an experiment can vary. A typical yield from an unstimulated mouse is in the range of 3-5 x 105 alveolar macrophages, however this number can vary greatly based on factors such as size of the mouse, experience of the person performing the lavage, and the number of instillations of lavage fluid and the volume withdrawn from lungs. In this protocol lung lavage is performed with a closed thoracic cavity which along with repeated 1 ml lavages helps to increase cell yield.
<ol>
	<li>Euthanize mouse by administering a lethal dose of avertin anesthetic (12.5 mg) intraperitoneally (i.p.) using aseptic conditions to keep the solution sterile.</li>
	<li>Spray mouse thoroughly with 70% ethanol.</li>
	<li>Remove skin from the cranial end of animal exposing the thorax (Figure 1A).
	<ol>
		<li>Make a small cut in skin just beneath the diaphragm.</li>
		<li>Insert finger into the cut and pull skin away from the cranial end of the animal (Figure 1A).</li>
		<li>Push up slightly on the head, extending the ventral side of the neck (Figure 1A).</li>
	</ol>
	</li>
	<li>Cut a small hole through the rib cage into the right side of the thorax to deflate the lungs and also allow visualization of lungs during the following steps.</li>
	<li>Locate the trachea, and carefully dissect away surrounding tissue.
	<ol>
		<li>Remove the salivary glands, sternohyoid muscles and larynx, etc. with curved forceps.</li>
		<li>Carefully lift with a curved forceps and cut away with scissors the thin tissue covering the trachea (adventitia) revealing the underlying ringed cartilage structure.</li>
	</ol>
	</li>
	<li>Insert catheter into trachea.
	<ol>
		<li>Using curved forceps, place a suture behind (dorsal to) the trachea and pull a 10 cm length through the opening.</li>
		<li>Starting above the cricoid (the thickened ring of cartilage) carefully guide a 22 G catheter down the trachea stopping where the trachea disappears behind the sternum.</li>
		<li>Firmly tie suture around trachea and catheter for a snug fit. Remove needle from catheter.</li>
	</ol>
	</li>
	<li>Assemble 4-way stopcock.
	<ol>
		<li>Fill one 6 ml syringe with DPBS, 1% FBS and attach to 4-way stopcock as indicated in Figure 1B.</li>
		<li>Attach an empty 6 ml syringe at the indicated position on the 4-way stopcock (Figure 1B).</li>
		<li>Attach open port on the 4-way stopcock to catheter (Figure 1B). Be careful to not dislodge catheter from trachea.</li>
	</ol>
	</li>
	<li>Collect lavage fluid from lungs.
	 Note: Investigators may opt to use lower volumes of lavage fluid to minimize the possibility of damage to the tissue. It is important that the same approach be consistently applied in all mouse groups.
	<ol>
		<li>Turn the handle on the stopcock so the empty syringe is closed and slowly inject ~1 ml DPBS, 1% FBS to inflate lungs. Observe lung inflation through the hole cut in step 2.4.</li>
		<li>Be careful NOT to overinflate the lungs.</li>
		<li>Turn the handle on the stopcock so that the full syringe is closed, and carefully withdraw the fluid from the lungs.</li>
		<li>Observe the lungs deflate through the hole cut in step 1.4. The catheter may need to be moved slightly back or forth to get a good draw. Take special precaution to keep the catheter within the trachea.</li>
		<li>Repeat steps 2.8.1-2.8.4 5-6x until all the DPBS, 1% FBS has been injected and withdrawn from the lungs. Note: recovery of lung fluid will not be 100% of injected fluid.</li>
	</ol>
	</li>
	<li>Empty syringe with lavage fluid into a 50 ml conical tube. Keep cells at room temperature.</li>
	<li>Pellet cells by centrifuging at 300 x g for 5 min at room temperature and pour off supernatant. This can be saved for cytokine analysis if desired.</li>
	<li>Resuspend cells in 1-5 ml RPMI 1640 with 10% FBS and count on a hemocytometer. In the unstimulated mouse, &#62; 95% of cells harvested by BAL are expected to be macrophages, which can be confirmed by cytology.</li>
</ol>
3. Culturing and Harvesting Fungus
The Aspergillus fumigatus used in this protocol expresses GFP throughout all stages of its life cycle: spores, germlings and hyphae. This fungal strain was provided by Dr. Margo Moore, Simon Fraser University, Burnaby, BC, Canada.
<ol>
	<li>Plate conidia of an Aspergillus fumigatus strain expressing GFP on Sabouraud brain heart infusion (BHI) agar slants with Chloramphenicol (0.05 g/L) and Gentamicin (0.5 g/L).</li>
	<li>Incubate in the dark loosely capped for 7 to 10 days at room temperature.</li>
	<li>Harvest conidia by washing slant with 10 ml of 0.01% Tween 20 in DPBS. Lightly scrape the fungus with the stripette to loosen. Rinse the slant several times to increase yield.</li>
	<li>Pass conidial suspension through a 100 &#181;m cell strainer into a 50 ml conical.</li>
	<li>Pellet conidia by centrifuging at 400 x g for 10 min.</li>
	<li>Resuspend conidia in 50 ml DPBS and count using a hemocytometer.</li>
	<li>Wash twice with DPBS and dilute to desired concentration.</li>
</ol>
4. Fungal Challenge and Confocal Microscopy
To investigate the role of macrophage NADPH oxidase in host defense, the ability of isolated alveolar macrophages from mice of three genotypes to control the growth of phagocytosed Aspergillus spores was evaluated. The genotypes used in this study include; 1) wild-type mice, 2) Ncf1*/* Mn- mice with a naturally occurring Ncf1 mutation (Ncf1 encodes for p47phox, a required component of the phagocyte NADPH oxidase) leaving them NADPH oxidase deficient, and 3) Ncf1*/* Mn+ transgenic mice (MN denotes the transgene harboring wildtype Ncf1 under the control of the human CD68 promoter) where NADPH oxidase activity has been reconstituted in the monocyte/macrophage population10,11.
Confocal microscopy is best suited for this assay due to the presence and growth of non-phagocytosed conidia on the slides. The confocal microscope will allow for visualization of individual planes within the Z-axis, and enable differentiation between fungus growing around rather than inside macrophage. All confocal images are acquired using a 63X oil immersion lens. Z-stacks are acquired with an electronic zoom factor of 2.5. The thickness of a Z-stack is determined using DAPI and bright field images to locate the top and bottom of the field. Z-stacks ranged from 14 to 24 &#181;m with individual slices ~1 &#181;m thick. For quantification of macrophages, single scans of 1X electronic zoom were performed on 10 fields per genotype. Intact macrophages were identified based on PKH26 and DAPI staining of the membrane and nucleus respectively. Conidia were identified based on FITC expression and bright field image.
<ol>
	<li>Prepare sterile coverslips and plates
	<ol>
		<li>Under a Biological Safety Cabinet, soak 22 x 22 mm glass coverslips in 70% ethanol for 5-10 min.</li>
		<li>Rinse coverslips in sterile PBS.</li>
		<li>Using sterile forceps, gently place a single coverslip in the bottom of each well of a sterile singly packed 6-well plate.</li>
	</ol>
	</li>
	<li>Seed ~1 x 106 harvested PKH26 labeled alveolar macrophages suspended in 300 &#181;l RPMI 1640, 10% FBS onto each coverslip. The exact number may vary depending on the harvest yield.</li>
	<li>Allow macrophage to adhere by incubating at 37 &#176;C with 5% CO2 overnight.</li>
	<li>The following morning, add ~1 x 107 GFP-expressing A. fumigatus conidia suspended in 100 &#181;l pre-warmed (37 &#176;C) RPMI 1640, 10% FBS.</li>
	<li>Return plate to 37 &#176;C incubator with 5% CO2.</li>
	<li>Cells should be fixed for a minimum of 2 hr at room temperature or overnight at 4 &#176;C. At 3, 7, and 14 hr fix cells by adding an equal volume of 2% formaldehyde to each well (final concentration 1% formaldehyde). Place plates for the early time points at 4 &#176;C overnight. For the final time point (14 hr), fix cells at room temperature for 2 hr.</li>
	<li>After fixation, gently remove coverslips using forceps and rinse in DPBS.</li>
	<li>Remove excess fluid by placing the edge of the coverslip on a folded Kimwipe.</li>
	<li>Apply 6 &#181;l fluorescence mounting medium with DAPI to a glass microscope slide.</li>
	<li>Lay one edge of the coverslip on the slide and gently drop with cell side down into the mounting medium. Mounting medium will spread and form an even layer beneath the coverslip. There is no need to press on the coverslip.</li>
	<li>Seal edges of coverslip with clear nail polish and allow to air dry.</li>
	<li>Store prepared slides in a slide box (protected from light) at room temperature until ready to analyze by confocal microscopy.</li>
</ol>

<ol>
	<li>Reeves, E. P., 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=Killing+activity+of+neutrophils+is+mediated+through+activation+of+proteases+by+K++flux.">Killing activity of neutrophils is mediated through activation of proteases by K+ flux.</a> Nature. 416, 291-297 (2002).</li><li>Bianchi, M., 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=Restoration+of+NET+formation+by+gene+therapy+in+CGD+controls+aspergillosis.">Restoration of NET formation by gene therapy in CGD controls aspergillosis.</a> Blood. 114, 2619-2622 (2009).</li><li>Vethanayagam, R. 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+NADPH+oxidase+versus+neutrophil+proteases+in+antimicrobial+host+defense.">Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense.</a> PLoS One. 6, (2011).</li><li>Grimm, M. J., 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=Monocyte-+and+Macrophage-Targeted+NADPH+Oxidase+Mediates+Antifungal+Host+Defense+and+Regulation+of+Acute+Inflammation+in+Mice.">Monocyte- and Macrophage-Targeted NADPH Oxidase Mediates Antifungal Host Defense and Regulation of Acute Inflammation in Mice.</a> The Journal of Immunology. 190, 4175-4184 (2013).</li><li>Schaffner, A., Douglas, H., Braude, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+protection+against+conidia+by+mononuclear+and+against+mycelia+by+polymorphonuclear+phagocytes+in+resistance+to+Aspergillus.+Observations+on+these+two+lines+of+defense+in+vivo+and+in+vitro+with+human+and+mouse+phagocytes.">Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes.</a> J Clin Invest. 69, 617-631 (1982).</li><li>Philippe, B., 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=Killing+of+Aspergillus+fumigatus+by+alveolar+macrophages+is+mediated+by+reactive+oxidant+intermediates.">Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates.</a> Infect Immun. 71, 3034-3042 (2003).</li><li>Cornish, E. J., 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=Reduced+nicotinamide+adenine+dinucleotide+phosphate+oxidase-independent+resistance+to+Aspergillus+fumigatus+in+alveolar+macrophages.">Reduced nicotinamide adenine dinucleotide phosphate oxidase-independent resistance to Aspergillus fumigatus in alveolar macrophages.</a> J Immunol. 180, 6854-6867 (2008).</li><li>Jennings, J. H., Linderman, D. J., Hu, B., Sonstein, J., Curtis, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytes+recruited+to+the+lungs+of+mice+during+immune+inflammation+ingest+apoptotic+cells+poorly.">Monocytes recruited to the lungs of mice during immune inflammation ingest apoptotic cells poorly.</a> Am J Respir Cell Mol Biol. 32, 108-117 (2005).</li><li>Davidson, B. A., 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=DISCRIMINATION+OF+RESIDENT+AND+INFILTRATED+ALVEOLAR+MACROPHAGES+BY+FLOW+CYTOMETRY+IN+INFLUENZA+A+VIRUS-INFECTED+MICE.">DISCRIMINATION OF RESIDENT AND INFILTRATED ALVEOLAR MACROPHAGES BY FLOW CYTOMETRY IN INFLUENZA A VIRUS-INFECTED MICE.</a> Experimental Lung Research. 31, 323-339 (2005).</li><li>Gelderman, K. A., 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=Macrophages+suppress+T+cell+responses+and+arthritis+development+in+mice+by+producing+reactive+oxygen+species.">Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species.</a> J Clin Invest. 117, 3020-3028 (2007).</li><li>Pizzolla, A., 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=Reactive+oxygen+species+produced+by+the+NADPH+oxidase+2+complex+in+monocytes+protect+mice+from+bacterial+infections.">Reactive oxygen species produced by the NADPH oxidase 2 complex in monocytes protect mice from bacterial infections.</a> J Immunol. 188, 5003-5011 (2012).</li><li>Luther, K., 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=Characterisation+of+the+phagocytic+uptake+of+Aspergillus+fumigatus+conidia+by+macrophages.">Characterisation of the phagocytic uptake of Aspergillus fumigatus conidia by macrophages.</a> Microbes and Infection. 10, 175-184 (2008).</li><li>Geunes-Boyer, 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=Surfactant+Protein+D+Increases+Phagocytosis+of+Hypocapsular+Cryptococcus+neoformans+by+Murine+Macrophages+and+Enhances+Fungal+Survival.">Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival.</a> Infection and Immunity. 77, 2783-2794 (2009).</li><li>Garc&#237;a-Rodas, R., Gonz&#225;lez-Camacho, F., Rodr&#237;guez-Tudela, J. L., Cuenca-Estrella, M., Zaragoza, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Interaction+between+Candida+krusei+and+Murine+Macrophages+Results+in+Multiple+Outcomes,+Including+Intracellular+Survival+and+Escape+from+Killing.">The Interaction between Candida krusei and Murine Macrophages Results in Multiple Outcomes, Including Intracellular Survival and Escape from Killing.</a> Infection and Immunity. 79, 2136-2144 (2011).</li><li>Ryan, L. K., Vermeulen, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+macrophages+from+C3H/HeJ+mice+show+sensitivity+to+endotoxin.">Alveolar macrophages from C3H/HeJ mice show sensitivity to endotoxin.</a> Am J Respir Cell Mol Biol. 12, 540-546 (1995).</li><li>Redente, E. F., 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=Differential+polarization+of+alveolar+macrophages+and+bone+marrow-derived+monocytes+following+chemically+and+pathogen-induced+chronic+lung+inflammation.">Differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogen-induced chronic lung inflammation.</a> J Leukoc Biol. 88, 159-168 (2010).</li><li>Schaffner, A., Douglas, H., Braude, A. I., Davis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+of+Aspergillus+spores+depends+on+the+anatomical+source+of+the+macrophage.">Killing of Aspergillus spores depends on the anatomical source of the macrophage.</a> Infect Immun. 42, 1109-1115 (1983).</li><li>Goodridge, H. 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The authors have nothing to disclose.

Using this approach for ex vivo analysis of the antifungal activity of macrophages together with in vivo fungal challenge, we previously demonstrated that NADPH oxidase in macrophages plays an important role in defense against A. fumigatus4. The use of isolated alveolar macrophages enables us to focus on their antifungal activity in the absence of other immune cells (e.g.,&#160;recruited neutrophils) that defend against Aspergillus in vivo.
Various visualization strategies have been used to evaluate macrophage antifungal activity in vivo and ex vivo. Luther et al., used confocal microscopy to study phagocytosis of Aspergillus conidia by human (harvested from lung transplant recipients) and mouse AMs12. Geunes-Boyer et al. characterized the interaction between Candida krusei and murine macrophages following phagocytosis. They used a macrophage cell line, RAW264.7 and primary macrophage from the peritoneum13. Garcia-Rodas et al. showed that surfactant protein-D binds to and facilitates the phagocytosis and survival of Cryptococcus neoformans14. SP-D binding was also investigated in vivo using SP-D-/- mice intranasally inoculated with Alexa Fluor 488-labeled C. neoformans. Following lung lavage, AMs were immunofluorescently labeled and confocal microscopy was used to analyze phagocytosis.
Macrophages from different anatomic sites (e.g.&#160;alveolar, splenic, peritoneal), generated with different stimuli (e.g.,&#160;thioglycollate elicitation or in vitro differentiation), or from cell lines (e.g.,&#160;RAW264.7) can have dramatically different antimicrobial and inflammatory responses15-18. Therefore, to most closely model the host response to inhaled fungi, we used alveolar macrophages harvested by bronchoalveolar lavage from unstimulated mice immediately following sacrifice.
Initially we used live-cell imaging to evaluate macrophage antifungal activity4. Alveolar macrophage were plated in chambered glass slides then seeded with GFP expressing Aspergillus. After 3 hr, non-phagocytosed conidia were removed by gentle washing to allow unobstructed visualization of phagocytosed conidia. Conidial germination was then monitored by time-lapse imaging at 30 min intervals using bright light images to visualize macrophage and GFP fluorescence to visualize fungi. This method has the advantage of acquiring sequential images of the same primary cell culture. However, the tendency for the macrophage to migrate across the field of view during the course of acquisition allowed for a qualitative rather than a quantitative assessment of conidiocidal activity. Another disadvantage of live-cell imaging is the lack of clarity and detail as cells move in and out of the plane of focus.
To obtain images with greater detail we chose confocal microscopy which enables the reconstruction of three-dimensional structures from the obtained images. Three-dimensional reconstruction gives the ability to differentiate between hyphae growing around rather than in or through a macrophage. With this ability to view the cells and fungus in three-dimensions, removal of non-phagocytosed conidia at 3 hr is no longer a necessary step. Conidia can also be precisely distinguished as intracellular versus extracellular.
For enhanced visualization, alveolar macrophage were labeled in vivo with PKH26. This fluorescent dye has a long aliphatic tail which stably incorporates into the lipid regions of cell membranes. When injected intravenously, this lipophilic dye is taken up by phagocytic cells both in the bloodstream and in tissues. After 10 days, labeled phagocytes are cleared from murine circulation and remain only in the tissues9, allowing for the collection of already labeled alveolar macrophage by BAL. With the use of in vivo labeling, post-harvest manipulation of alveolar macrophages is minimized.
Another advantage to this protocol is the use of a GFP-expressing A. fumigatus strain. This fungus expresses GFP from the conidial stage through the hyphal stage and has no need for further staining. With dual staining of macrophages and Aspergillus, we are able to identify phagocytosed conidia.
A limiting factor in this protocol is the macrophage yield from the BAL. A typical yield from an unstimulated mouse is in the range of 3-5 x 105 alveolar macrophages, however this number can vary greatly based on factors such as size of the mouse, experience of the person performing the lavage, and the number of instillations of lavage fluid and the volume withdrawn from lungs. In this protocol, lung lavage is performed with a closed thoracic cavity which along with repeated 1 ml instillations helps to increase cell yield when compared to an open thoracic cavity and fewer instillations. Mouse numbers can also be increased to increase harvest yield.

Alveolar macrophages are the first-line phagocytic cells that encounter inhaled microbes. Macrophages recognize Aspergillus motifs by pathogen recognition receptors, ingest and limit the growth of inhaled spores (conidia), and initiate inflammatory responses. The phagocyte NADPH oxidase converts molecular oxygen to superoxide anion and downstream reactive oxygen intermediates (ROIs). Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe bacterial and fungal infections and by excessive inflammatory responses.
Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. Prior studies have shown that alveolar macrophages ingest and kill Aspergillus spores, whereas neutrophils principally target the hyphal stage5. However, there have been conflicting results as to the role of NADPH oxidase in macrophage in controlling the growth of A. fumigatus spores6,7.
The goal of this method was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus spores. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. To most closely model the host response to inhaled fungi, we used alveolar macrophages harvested by bronchoalveolar lavage from unstimulated mice immediately following sacrifice. The use of isolated alveolar macrophages enabled us to focus on their antifungal activity in the absence of other immune cells (e.g.,&#160;recruited neutrophils) that defend against Aspergillus in vivo. In this method, alveolar macrophages were stained in vivo prior to harvest so as to minimize the amount of post-harvest manipulation.
Another advantage to this protocol is the use of a GFP-expressing A. fumigatus strain. This fungus expresses GFP from the conidial stage through the hyphal stage and has no need for further staining. To obtain images with greater detail, we chose confocal microscopy which enables the reconstruction of three-dimensional structures from the obtained images. Three-dimensional reconstruction gives the ability to differentiate between hyphae growing around rather than in or through a macrophage. Conidia can also be precisely distinguished as intracellular versus extracellular.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2,2 Tribromoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T48402</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methyl-2-butanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A-1685</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (without calcium and magnesium)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4CL</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A0171</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P91444</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">22 gauge X 1.00 inch i.v. catheter</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">381523</td>
			<td>Insyte Autoguard Winged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">4-way stopcock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-700-077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">suture thread</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 with L-glutamine</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH30396.03</td>
			<td>heat inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hema 3 Stain Set</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-122-911</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytoseal 60</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">8310-4</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">297252</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 micron Cell Strainers</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">64753-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scissors</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sorval ST40 Centrifuge with swing bucket rotor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytospin 2 centrifuge</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Cell Culture Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>37 &#176;C, 5%&#160; CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">22X22 mm micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366-227</td>
			<td>glass coverslips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 well Cell Culture Plates</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Neutral Buffered Formalin</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH0502-1LP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vectashield Mounting medium with DAPI</td>
			<td style="width:71px;">Vector Laboratories</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica TCS SP2 system with a laser point scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Mounted on DMIRE2 fluorescence microscope</td>
		</tr>
	</tbody>
</table>

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.

Collecting alveolar macrophage by BAL from unstimulated mice will result in a &#62; 95% pure population based on cytology. The 3 and 7 hr (Figure 2) time points precede the hyphal stage of fungal growth and allow for a comparison of phagocytic efficiency of conidia among genotypes. The 14 hr time point is where genotypes can be compared with regard to the ability to inhibit the transition of phagocytosed conidia to the tissue-invasive hyphal stage (Figure 3). Intact macrophage can be identified by confocal microscopy based on PKH26 and DAPI staining of the membrane and nucleus, respectively (Figures 2&#160;and&#160;3). Conidia and hyphae are identified based on GFP expression (Figures 2 and 3).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51678/51678fig1highres.jpg" src="/files/ftp_upload/51678/51678fig1.jpg" /> 
Figure 1.&#160;Anatomical Directional References and the 4-Way Stopcock. A) The anatomical directional references for a mouse are shown. Cranial is in the direction of the head, while caudal is in the direction of the tail. The ventral side of a quadruped is toward the belly or the lower surface of the animal, while the dorsal side is toward the back or the upper surface of the animal. B) The 4-way stopcock has three luer connections and a handle that rotates 360&#176;&#160;to direct the flow of fluids through the stopcock. Placement of the empty syringe, the full syringe and the catheter are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51678/51678fig1highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51678/51678fig2highres.jpg" src="/files/ftp_upload/51678/51678fig2.jpg" /> 
Figure 2.&#160;Phagocytosis of A. fumigatus spores is similar between genotypes at 7 hr. To evaluate the ability of isolated alveolar macrophages with NADPH oxidase activity to limit the growth of phagocytosed conidia, (A)&#160;wild-type, (B)&#160;Ncf1*/*Mn+, and (C)&#160;Ncf1*/*Mn- mice were administered i.v. PKH26. Alveolar macrophages were harvested by BAL 10 days later and seeded with conidia of GFP-expressing A. fumigatus. Cells were fixed at 3, 7, and 14 hr after addition of conidia. At 3 (data not shown) and 7 hr both the number of total macrophages and macrophage with &#8805; 1 phagocytosed conidia (arrows) were similar among the three genotypes, reflecting similar phagocytosis efficiency. Intact macrophages are identified based on PKH26 (red) and DAPI (blue) staining of membrane and nuclei respectively. The fluorescence images from a single Z-plane are overlaid with the bright field image. The bright field image consists of light transmitted through the entire specimen and so when overlain on the fluorescent Z-plain allows visualization of cells and conidia within the entire Z-stack. Scale bar, 19 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51678/51678fig2highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51678/51678fig3highres.jpg" src="/files/ftp_upload/51678/51678fig3.jpg" /> 
Figure 3. Macrophage NADPH oxidase is required to control the growth of A. fumigatus spores. Confocal microscopy of (A, D)&#160;wild-type, (B, E)&#160;Ncf1*/*Mn+, and (C, F)&#160;Ncf1*/*Mn- alveolar macrophage at 14 hr after seeding with GFP+ A. fumigatus conidia. Intact macrophages are identified based on PKH26 (red) and DAPI (blue) staining of membranes and nuclei, respectively. Solid arrows show intact macrophages with at least one phagocytosed conidia and hollow arrows point to hyphae. Numerous AMs with at least 1 phagocytosed conidia were observed with wild-type and Ncf1*/*Mn+ cells, but not with NADPH oxidase-deficient cells. GFP signal in hyphae was variable based on the plane of the z-stacked image shown. A-C) Fluorescence images from a single Z-plane with the bright field image enable visualization of cells and hyphae within the entire Z-stack. D-F) Fluorescence images from a single Z-plane without the bright field background enable identification of cells, but not the spatial relationship of cells and fungi in different Z-planes. Scale bar, 19 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51678/51678fig3highres.jpg" target="_blank">Click here to view larger image.</a>

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Assessing Anti-fungal Activity of Isolated Alveolar Macrophages 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 ...

assessing-anti-fungal-activity-isolated-alveolar-macrophages-confocal

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Roswell Park Cancer Institute. A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

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

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

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. ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

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 ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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 ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.