Name: assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
Uploaded: 2014-07-09T21:30:00
Description: Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

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...

<ol>
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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.
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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.

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			<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>
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		<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>
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		<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<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>
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			<td height="21" style="height:21px;width:216px;">suture thread</td>
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			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">scissors</td>
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			<td height="21" style="height:21px;width:216px;">forceps</td>
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			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
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			<td style="width:119px;"></td>
			<td></td>
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		<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>
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			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
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			<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>
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		<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>
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		<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 ide...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

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 ...

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