Name: Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy (Video) | JoVE Journal
Uploaded: 2014-07-09T21:30:00.000+00:00
Duration: 9 min 4 s
Description: Learn to assess anti-fungal activity of isolated mouse alveolar macrophages using confocal microscopy. This method evaluates phagocytic control of Aspergillus spores for immunology research.

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5 minutes

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/pubmed?term=((Killing+activity+of+neutrophils+is+mediated+through+activation+of+proteases+by+K%2B+flux%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Restoration+of+NET+formation+by+gene+therapy+in+CGD+controls+aspergillosis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Role+of+NADPH+oxidase+versus+neutrophil+proteases+in+antimicrobial+host+defense%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Monocyte-+and+Macrophage-Targeted+NADPH+Oxidase+Mediates+Antifungal+Host+Defense+and+Regulation+of+Acute+Inflammation+in+Mice%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((Killing+of+Aspergillus+fumigatus+by+alveolar+macrophages+is+mediated+by+reactive+oxidant+intermediates%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">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/pubmed?term=((Reduced+nicotinamide+adenine+dinucleotide+phosphate+oxidase-independent+resistance+to+Aspergillus+fumigatus+in+alveolar+macrophages%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Monocytes+recruited+to+the+lungs+of+mice+during+immune+inflammation+ingest+apoptotic+cells+poorly%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((DISCRIMINATION+OF+RESIDENT+AND+INFILTRATED+ALVEOLAR+MACROPHAGES+BY+FLOW+CYTOMETRY+IN+INFLUENZA+A+VIRUS-INFECTED+MICE%5BTitle%5D)+AND+%22Experimental+Lung+Research%22%5BJournal%5D)">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/pubmed?term=((Macrophages+suppress+T+cell+responses+and+arthritis+development+in+mice+by+producing+reactive+oxygen+species%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((Reactive+oxygen+species+produced+by+the+NADPH+oxidase+2+complex+in+monocytes+protect+mice+from+bacterial+infections%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Characterisation+of+the+phagocytic+uptake+of+Aspergillus+fumigatus+conidia+by+macrophages%5BTitle%5D)+AND+%22Microbes+and+Infection%22%5BJournal%5D)">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/pubmed?term=((Surfactant+Protein+D+Increases+Phagocytosis+of+Hypocapsular+Cryptococcus+neoformans+by+Murine+Macrophages+and+Enhances+Fungal+Survival%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">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>García-Rodas, R., González-Camacho, F., Rodríguez-Tudela, J. L., Cuenca-Estrella, M., Zaragoza, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Interaction+between+Candida+krusei+and+Murine+Macrophages+Results+in+Multiple+Outcomes%2C+Including+Intracellular+Survival+and+Escape+from+Killing%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">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/pubmed?term=((Alveolar+macrophages+from+C3H%2FHeJ+mice+show+sensitivity+to+endotoxin%5BTitle%5D)+AND+%22Am+J+Respir+Cell+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Differential+polarization+of+alveolar+macrophages+and+bone+marrow-derived+monocytes+following+chemically+and+pathogen-induced+chronic+lung+inflammation%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">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/pubmed?term=((Killing+of+Aspergillus+spores+depends+on+the+anatomical+source+of+the+macrophage%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Killing of Aspergillus spores depends on the anatomical source of the macrophage.</a> Infect Immun. 42, 1109-1115 (1983).</li>
<li>Goodridge, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+use+of+CARD9+by+dectin-1+in+macrophages+and+dendritic+cells%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Differential use of CARD9 by dectin-1 in macrophages and dendritic cells.</a> J Immunol. 182, 1146-1154 (2009).</li>
</ol>

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.
<p class="jo...

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><tbody><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma</td><td>PKH26GL-1KT</td><td></td></tr><tr><td>2,2,2 Tribromoethanol</td><td>Sigma</td><td>T48402</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma</td><td>A-1685</td><td>Used to make AvertinAnesthetic</td></tr><tr><td>Dulbecco&amp;#039;s Phosphate Buffered Saline (without calcium and magnesium)</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>A0171</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P91444</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td>Used to make ACK red cell lysis buffer</td></tr><tr><td>22 G x 1.00 inch i.v. catheter</td><td>BD</td><td>381523</td><td>Insyte Autoguard Winged</td></tr><tr><td>Insulin syringes</td><td></td><td></td><td></td></tr><tr><td>6 ml Syringes</td><td></td><td></td><td></td></tr><tr><td>4-way stopcock</td><td>Fisher Scientific</td><td>50-700-077</td><td></td></tr><tr><td>Suture thread</td><td></td><td></td><td></td></tr><tr><td>50 ml conical centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>gauze sponges (4 inch square)</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>heat inactivated</td></tr><tr><td>Hema 3 Stain Set</td><td>Fisher Scientific</td><td>22-122-911</td><td></td></tr><tr><td>Cytoseal 60</td><td>Thermo Scientific</td><td>8310-4</td><td></td></tr><tr><td>Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td><td>BD</td><td>297252</td><td></td></tr><tr><td>&lt;em&gt;Aspergillus fumigatous&lt;/em&gt; strain expressing green fluorescent protein</td><td></td><td></td><td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td></tr><tr><td>100 &amp;mu;m Cell strainers</td><td>BD Falcon</td><td>64753-00</td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Biological Safety Cabinet Class II</td><td></td><td></td><td></td></tr><tr><td>Sorval ST40 Centrifuge with swing bucket rotor</td><td></td><td></td><td></td></tr><tr><td>Leica DM1000 light microscope</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Cytospin 2 centrifuge</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td>37 &amp;deg;C, 5%&amp;nbsp; CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>22 x 22 mm micro cover glass</td><td>VWR</td><td>48366-227</td><td>glass coverslips</td></tr><tr><td>6-well Cell culture plates</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>VWR</td><td>BDH0502-1LP</td><td></td></tr><tr><td>Vectashield mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Leica TCS SP2 system with a laser point scanner</td><td></td><td></td><td>Mounted on DMIRE2 fluorescence microscope</td></tr></tbody></table>

assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

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 &gt; 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 iden...

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 ...

The overall goal of this procedure is to compare the ability of macrophages to control the growth of ceto fungal candia ex vivo. This is accomplished by first labeling the alveolar macrophages in vivo with a lipophilic dye PKH 26. The second step of the procedure is to harvest alveolar macrophages from the inoculated mice by bronchoalveolar lavage.The third step is to culture and collect GFP positive fungal spores. The final steps are to challenge macrophages with GFP positive candia and fixed cells at predetermined time points. Ultimately, with minimal ex vivo cell manipulation, confocal microscopy can reveal the ability of alveolar macrophages to control the growth of phagocytose.Spores First dilute the lipophilic dye P HK 26, 1 to five. In Diluent C, the dye will be taken up by the FOC acidic cells. Load 100 microliters of diluted P HK 26 into a one half cc syringe and inject the dye intravenously retroorbital, or by the tail vein.Estimate that each mouse will provide three to 500, 000 labeled macrophages after 10 days. Euthanize the mice and prepare to open the chest cavity from the diaphragm. Cut a small hole in the rib cage to the right of the thorax to deflate the lungs and for later viewing of the lungs inflation.Next, locate and isolate the trachea from the surrounding tissues. Then using curved forceps, carefully cut away the adventitia, which covered the trachea. Once exposed, insert a catheter into the trachea.It is very important to avoid puncturing the trachea. In executing this maneuver, secure the catheter with a 10 centimeter length of suture looped around the trachea. Then remove the needle from the catheter.Next, fill a six milliliter syringe with lavage fluid and attach it to a four-way stop cock. Attach an empty six milliliter syringe at another position and carefully connect the open port to the catheter. With the assembly in place, open the stop cock to the full syringe and slowly inject about one milliliter of solution into the lungs.Next, turn the stop cock to close the syringe and to withdraw fluid. Then carefully withdraw the macrophage rich lung fluid while observing the lungs deflate. Repeat this process until all the solution has been injected into and collected from the lungs.Expect some fluid loss. You'll not be able to remove the fluid from the lungs if the catheter is in the wrong position. Carefully move the catheter back and forth until the fluid can easily be withdrawn from the lungs.Be certain not to allow the catheter to fall out. Then transfer the collected lavage fluid into a 50 milliliter conical to collect the cells centrifuge the fluid at 300 Gs for five minutes. At room temperature, pour off the supernatant and if desired, save it for cytokine analysis.Resuspend the cells in one to five milliliters of RPMI 1640 with 10%FBS and determine their density. At least 95%of the cells should be macrophages to culture. Aspergillus fumigatus expressing GFP Berran brain heart infusion.A agri slants treated with 0.05 grams of chloramphenicol and 0.5 grams of gentamycin per liter. Incubate the fungus on the slants loosely capped in the dark and at room temperature. After seven to 10 days, harvest the canadia by washing each slant with 10 milliliters of 0.01%Tween 20 in DPBS use a STR PET to loosen the fungus and use several rinses to increase the yield.Pass the suspension through a 100 micron strainer and collect the filtrate containing the kedia. In a 50 milliliter conical pool the kedia with a 10 minute spin at 400 Gs at room temperature. Then resuspend them in 50 milliliters of DPBS and determine their concentration with a hemo cytometer.Lastly, wash the kedia twice more with DBBS and resuspend them at the desired concentration. First cover slips and plates must be prepared under a biological safety cabinet. Soak 22 millimeter square cover slips in 70%ethanol for five to 10 minutes.Then rinse them off in PBS for each time point to process, use forceps to gently place single sterilized cover slips into the wells of a six well plate load a well for each condition tested onto each cover slip seed about 1 million PKH 26 labeled macrophages in 300 microliters of RPMI 1640 with 10%FBS incubate the cells at 37 degrees Celsius with 5%carbon dioxide overnight. The following morning to each seated cover slip at 10 million GFP expressing aspergillus fuma Gattis kedia in 100 microliters of warm RPMI 1640 with 10%FBS. Return the plates to the incubator for the duration of the challenge and fix the cells at predetermined time intervals to score the results.To fix a plate of cells, add one volume of 2%formaldehyde to each well, and let the plate sit for two hours at room temperature. After two hours, move the plate to four degrees Celsius until the other plates have also been fixed. First, prepare a glass slide with six microliters of fluorescent mounting medium with dappy When all the plates are fixed, gently remove the cover slips with forceps and rinse them in a bath of DPBS for a minute.Then blot their edges to remove excess fluid. Next, gently lay the cover, slip down into the mounting medium prepared on the glass slide earlier. There is no need to press the slide for the medium to spread.Seal the slide with nail polish and allow it to air dry. Store the slides at room temperature in the dark until they can be viewed in vivo. Labeled alveolar.Macrophages were collected and then exposed to a fungal challenge. Wild type mice and mice carrying a mutation in N-A-D-P-H. Oxidase were used to harvest macrophages after three or seven hours.Phagocytosis was similar in the wild type and mutant strains. Cells containing candia positive for both PKH 26 and GFP were abundant in all three genotypes, however, at 14 hours differences emerged with respect to the ability of macrophages to control the growth of phagocytosed Candia. Many intact wild-type macrophages containing phagocytosed candia were found.This was also observed in cells with a functional N-A-D-P-H oxidase enzyme in the NCF one. Background by contrast, intact alveolar macrophages lacking N-A-D-P-H oxidase were scarce, suggesting that Phace toast Kedia had grown destroying the cells. This also suggests that controlling the growth of phace toast Kedia is N-A-D-P-H oxidase dependent Once mastered, this lavage technique can be done in about five minutes per mouse if performed properly.

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Learn to assess anti-fungal activity of isolated mouse alveolar macrophages using confocal microscopy. This method evaluates phagocytic control of Aspergillus spores for immunology research.

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

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