We thank Jeff Tucker, Erica Scappini, and Agnes Janoshazi for their help with microscopy, Ligon Perrow for her management of the mouse colony, and Jun Chen and Michael Sanderson for help with the tissue slicer, and Michael Fessler and Derek Cain for critical reading of the manuscript. This work was funded by the intramural branch of the NIEHS, NIH (ZIA ES102025-09), which is in turn sponsored by the Department of Health and Human Services.

Animal experimental procedures described in this paper were approved by the NIEHS Animal Care and Use Committee (IACUC).
1. Lung Preparation
<ol>
	<li>House mice between 6 and 12 weeks of age in specific pathogen-free conditions in accordance with the guidelines provided by the Institutional Animal Care and Use Committees. 
	NOTE: Imaged mice can be either na&#239;ve, or treated, depending on each researcher&#39;s specific interests. Here, we describe immune cell localization in na&#239...

<ol>
	<li>Schneider, T., van Velzen, D., Moqbel, R., Issekutz, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+quantitation+of+eosinophil+and+neutrophil+recruitment+to+allergic+lung+inflammation+in+a+brown+Norway+rat+model.">Kinetics and quantitation of eosinophil and neutrophil recruitment to allergic lung inflammation in a brown Norway rat model.</a> Am J Respir Cell Mol Biol. 17 (6), 702-712 (1997).</li><li>Vermaelen, K. Y., Carro-Muino, I., Lambrecht, B. N., Pauwels, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+migratory+dendritic+cells+rapidly+transport+antigen+from+the+airways+to+the+thoracic+lymph+nodes.">Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.</a> J Exp Med. 193 (1), 51-60 (2001).</li><li>Sung, S. 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=A+major+lung+CD103+(alphaE)-beta7+integrin-positive+epithelial+dendritic+cell+population+expressing+Langerin+and+tight+junction+proteins.">A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins.</a> J Immunol. 176 (4), 2161-2172 (2006).</li><li>Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lasker+Basic+Medical+Research+Award.+Dendritic+cells:+versatile+controllers+of+the+immune+system.">Lasker Basic Medical Research Award. Dendritic cells: versatile controllers of the immune system.</a> Nat Med. 13 (10), 1155-1159 (2007).</li><li>Jakubzick, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph-migrating,+tissue-derived+dendritic+cells+are+minor+constituents+within+steady-state+lymph+nodes.">Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes.</a> J Exp Med. 205 (12), 2839-2850 (2008).</li><li>Julia, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+restricted+subset+of+dendritic+cells+captures+airborne+antigens+and+remains+able+to+activate+specific+T+cells+long+after+antigen+exposure.">A restricted subset of dendritic cells captures airborne antigens and remains able to activate specific T cells long after antigen exposure.</a> Immunity. 16 (2), 271-283 (2002).</li><li>Nakano, H., 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=Migratory+properties+of+pulmonary+dendritic+cells+are+determined+by+their+developmental+lineage.">Migratory properties of pulmonary dendritic cells are determined by their developmental lineage.</a> Mucosal Immunol. 6 (4), 678-691 (2013).</li><li>Nakano, H., 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=Complement+receptor+C5aR1/CD88+and+dipeptidyl+peptidase-4/CD26+define+distinct+hematopoietic+lineages+of+dendritic+cells.">Complement receptor C5aR1/CD88 and dipeptidyl peptidase-4/CD26 define distinct hematopoietic lineages of dendritic cells.</a> J Immunol. 194 (8), 3808-3819 (2015).</li><li>Sanderson, M. 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J., Brendel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision-cut+tissue+slices:+applications+in+pharmacology+and+toxicology.">Precision-cut tissue slices: applications in pharmacology and toxicology.</a> Life Sci. 57 (21), 1887-1901 (1995).</li><li>Bergner, A., Sanderson, M. 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H., 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=Allergic+sensitization+through+the+airway+primes+Th17-dependent+neutrophilia+and+airway+hyperresponsiveness.">Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness.</a> Am J Respir Crit Care Med. 180 (8), 720-730 (2009).</li><li>Schlitzer, 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=Identification+of+cDC1-+and+cDC2-committed+DC+progenitors+reveals+early+lineage+priming+at+the+common+DC+progenitor+stage+in+the+bone+marrow.">Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.</a> Nat Immunol. 16 (7), 718-728 (2015).</li><li>Baluk, 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=Preferential+lymphatic+growth+in+bronchus-associated+lymphoid+tissue+in+sustained+lung+inflammation.">Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.</a> Am J Pathol. 184 (5), 1577-1592 (2014).</li><li>Jurisic, G., Iolyeva, M., Proulx, S. 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The protocol described here was originally developed to visualize the locations of two subsets of cDCs within the lung. However, this protocol can be readily adapted to study many different cell types, while maintaining cell viability and the three-dimensional architecture of the lung. The latter feature is an important advantage over cell culture systems and facilitates identification of rare cell types. The method relies on the generation of PCLS from the lung, and an appropriate combination of antibodies to identify s...

Following inhalation of pro-inflammatory stimuli such as lipopolysaccharide (LPS), there is a coordinated movement of immune cells into, within, and from the lung. For example, neutrophils are rapidly recruited to the lung parenchyma and airway. In addition, some professional antigen presenting cells known as conventional dendritic cells (cDCs) undergo a relatively complex migration pattern1,2. cDCs can be identified using flow cytometry, based in part on their display of the surface marker, CD11c. Distinct subsets of DCs can be distinguished by the differential surface expression of CD103 and CD11b3. Upon acquiring inhaled antigen, some cDCs exit the lung and migrate through the lymphatic vessels to lung-draining Lymph Nodes (LNs) where they present peptides to antigen-specific T cells4. This is a critical early event in the initiation of adaptive immune responses. For unknown reasons, however, not all cDCs that acquire inhaled antigens leave the lung, and many of these cells remain in that organ for several months5,6. This observation can be partly explained by the developmental ancestry of these cells because monocyte-derived CD11c+ cells lacking the chemokine receptor, CCR7, are unable to migrate to regional LNs7,8. It seems likely that the migration potential of cDCs is also determined, at least in part, by their anatomical position within the lung. However, the precise localization of these different populations of cDCs in the lung is not fully characterized. An improved knowledge of immune cell localization within the lung, and of the molecules that direct it, is needed for a better understanding of how the immune system of the lung becomes activated.
PCLS are being increasingly used as an ex vivo approach to visualize cellular positioning and cell-cell interactions, while maintaining the structural integrity of the lung architecture9,10. PCLS have been used to study lungs of many species, including mice, cattle, monkeys, sheep, horses, and humans11. A major advantage of this technique is that approximately 20 slices can be prepared from a single lobe of a mouse lung, thereby reducing the number of animals needed for individual experiments. Virtually all immune cell types, including DCs, macrophages, neutrophils, and T cells, are present in PCLS and maintain their normal structures.
PCLS can also be used to study calcium signaling and contractility of airway and smooth muscle cells after treatment with acetylcholine12 or methacholine13. In this approach, only a small portion of the lung is analyzed microscopically, but one study reported that measurements of airway contraction in PCLS vary only about 10% from slice to slice, and this variance is comparable to that seen using lung function tests in intact animals14. Other investigators have used PCLS as an ex vivo approach to study changes in cytokine expression and cell surface markers after incubation with LPS15. PCLS have also been used in an ex vivo model of hypoxic pulmonary vasoconstriction in small intra-acinar arteries. These vessels are located in the part of the lung that cannot be reached using other procedures, including recordings from dissected arterial segments or analysis of subpleural vessels16. Our lab has primarily used PCLS to visualize immune cell localization in live lung tissue at steady state and following an in vivo inflammatory stimulus. The procedures we have developed for this are as follows.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Prox1-TdTomato transgenic mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td>018128</td>
			<td>B6;129S-Tg(Prox1-tdTomato)12Nrud/J</td>
		</tr>
		<tr>
			<td>OT-II OVA-specific TCR x Nur77-GFP transgenic mice</td>
			<td>Jackson Laboratory</td>
			<td>004194, 006617</td>
			<td>B6.Cg-Tg(TcraTcrb)425Cbn/J x C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J&#160;</td>
		</tr>
		<tr>
			<td>Rag1 knock-out mice</td>
			<td>Jackson Laboratory</td>
			<td>002216</td>
			<td>B6.129S7-Rag1tm1Mom/J</td>
		</tr>
		<tr>
			<td>Ovalbumin, Low Endo, Purified</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS003059</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli</td>
			<td>Sigma-Aldrich Co.</td>
			<td>L2630-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing (Non-Sterile) 100 ft</td>
			<td>BD Diagnostic Systems</td>
			<td>427421</td>
			<td>0.86 mm inside diameter, 1.27 mm outside diameter</td>
		</tr>
		<tr>
			<td>GeneMate Sieve GQA Low Melt Agarose</td>
			<td>BioExpress</td>
			<td>E-3112-125</td>
			<td>2% solution dissolved in PBS at 70 &#176;C and held at 40 &#176;C.</td>
		</tr>
		<tr>
			<td>Compresstome VF-300</td>
			<td>Precisionary Instruments, Inc.</td>
			<td>VF-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Edge Stainless Razor Blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td>Disposable; 250/box. Blade should be changed for every lung.</td>
		</tr>
		<tr>
			<td>Krazy Glue All Purpose Instant Gel</td>
			<td>VWR</td>
			<td>500033-484</td>
			<td>Commonly available for $3/tube in local drugstores&#160;</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rat Serum</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>012-000-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse Serum</td>
			<td>Jackson ImmunoResearch Inc.</td>
			<td>015-000-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Buffer</td>
			<td>Made in House</td>
			<td>N/A</td>
			<td>PBS w/ 0.5% bovine serum albumin, 0.1% NaN3, pH 7.4</td>
		</tr>
		<tr>
			<td>Fc Blocker (anti-CD16/32 antibodies)</td>
			<td>Made in House</td>
			<td>N/A</td>
			<td>Supernatant of cultured hybridoma cell line 2.4G2</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b eFluor 450</td>
			<td>eBioscience</td>
			<td>48-0112-80</td>
			<td>Anti-mouse CD11b eFluor 450 (clone: M1/70)&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c Brilliant Violet 605</td>
			<td>BioLegend</td>
			<td>101237</td>
			<td>Brilliant Violet 605 anti-mouse CD11c (clone: M1/70)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c Phycoerythrin</td>
			<td>eBioscience</td>
			<td>12-0114-82</td>
			<td>PE conjugated anti-mouse CD11c (clone: N418)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c Allophycocyanin</td>
			<td>BD Phamingen</td>
			<td>550261</td>
			<td>APC-labeled anti-mouse CD11c 9clone: HL3)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD88 Phycoerythrin</td>
			<td>BioLegend</td>
			<td>135806</td>
			<td>PE anti-mouse CD88 (clone: 20/70)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD103 Allophycocyanin</td>
			<td>eBioscience</td>
			<td>17-1031-82</td>
			<td>Anti-mouse CD103 APC (clone: 2E7)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD90.2/Thy1.2 eF450</td>
			<td>eBioscience</td>
			<td>48-0902-82</td>
			<td>Anti-mouse CD90.2 eFluor 450 (clone: 53-2.1)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD172a/Sirp1a Allophycocyanin</td>
			<td>eBioscience</td>
			<td>17-1721-82</td>
			<td>Anti-mouse CD172a APC (clone: P84)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD324 Brilliant Violet 421</td>
			<td>BD Horizon</td>
			<td>564188</td>
			<td>BV421 mouse anti-E Cadherin (clone: 5E8 also known as 5E8-G9-B4)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD324 Alexa Fluor 488</td>
			<td>eBioscience</td>
			<td>53-3249-82</td>
			<td>Anti-CD324(E-Cadherin) Alexa Flour 488 (clone: DECMA-1)</td>
		</tr>
		<tr>
			<td>Anti-mouse CD324 Alexa Fluor 647</td>
			<td>eBioscience</td>
			<td>51-3249-82</td>
			<td>Anti-CD324(E-Cadherin) Alexa Flour 647 (clone: DECMA-1)</td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes 35mm petri dish, 14mm Microwell, No. 1.5 coverglass</td>
			<td>MatTek Corperation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered Coverglass</td>
			<td>ThermoFisher Scientific</td>
			<td>155411PK&#160;</td>
			<td>Pack of 16</td>
		</tr>
		<tr>
			<td>15 mm Coverslip, No. 1.5 Glass Thickness</td>
			<td>MatTek Corperation</td>
			<td>PCS-1.5-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Bare Platinum Wire</td>
			<td>World Precision Instruments</td>
			<td>PTP201</td>
			<td>0.020&#34; (0.5mm) diameter cut into ~1 cm long pieces and bent into an &#34;L&#34; shape</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36934</td>
			<td>Keep at 4 &#176;C, warm to room tempterature before use.</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, *LDEV-Free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss 880 multi-photon laser-scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Zen Black software version 8.1, 2012 (Zeiss)</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 20x/0.8 M27 objective lends</td>
			<td>Carl Zeiss</td>
			<td>420650-9901-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

precision-cut mouse lung slices to visualize live pulmonary dendritic cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

To identify the location of two DC subsets, CD11bhi cDCs and CD103+ cDCs, PCLS from C57BL/6 mice were cut and stained with monoclonal antibodies (mAbs) specific to CD11c, CD88, CD103, and CD324 (E-cadherin). Antibodies to CD324 stain airway epithelial cells, and CD88 is displayed on macrophages and neutrophils, but not cDCs8. This allowed us to distinguish cDCs from CD11c+ macrophages, and to observe the spatial relationship of each...

Watch this Scientific Journal Video about Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells at JoVE.com

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

The overall goal of this method is to use precision-cut lung slices, or PCLS, to visualize the localization and trafficking of various immune cell types in the lung. This method can help answer key questions in the immunology field, such as the localization and migration patterns of immune cells in the lung. The main advantage of this technique is that several interactions can be visualized in the live ex vivo tissue that maintains the three dimensional architecture of the lung.Though this method can provide insight into the localization and trafficking of immune cells during inflammation, it can also be used to visualize airway and smooth muscle tissue contraction. To harvest the lung tissue, first use scissors to open the skin and peritoneum from the middle of the abdomen up to the jaw of a six to 12 week old mouse, and move the intestines to the side of the abdominal cavity. Snip the inferior vena cava to drain the blood away from the lungs, and use the sharp tip of the scissors to puncture the diaphragm to allow expansion of the ribcage.Next, use forceps to manually pull the salivary glands and other tissues away from the trachea, and use fine forceps to make an incision in the trachea on the anterior side of the thickest band of cartilage, just large enough to allow a 20 gauge needle to penetrate. Now slide a 1.5 inch 20 gauge needle onto a section of polyethylene tubing, and cut the tubing at an approximately 45 degree angle. Attach the needle to a one milliliter syringe, and draw 0.8 milliliters of 40 degrees Celsius 2%low melting point agarose into the syringe.When all of the agarose has been loaded, insert the beveled end of the tubing into the incision, and slowly inject the agarose into the lungs. Without moving the needle or the syringe, tape the syringe to the polystyrene base to secure the needle within the trachea, and place the animal in 4 degrees Celsius storage for at least 10 minutes. Once the agarose has solidified, use scissors to carefully excise the lungs, and place them in a three centimeter dish containing ice cold PBS.Then turn the lobe for tissue slicing so that the interior section of the lung that connects to the trachea faces down. To slice the lung tissue, first place a small drop of All Purpose No Run gel superglue onto a metal syringe plunger, spreading the glue in a circular motion. When all of the glue has been applied, use forceps to immediately but gently grasp the excised lobe trachea side down, and dab the lobe on a tissue to remove the excess liquid.Carefully place the dried lobe on top of the plunger, and trim any extra tissue extending beyond the edge of the plunger. Move the plunger down so that the sides of the metal syringe move up and over the tissue, creating a well with the lung at the bottom of the syringe shaft that is no more than several centimeters deep. Tape around the bottom of the syringe to hold the plunger in place.Then carefully pour 40 degrees Celsius 2%low melt agarose into the syringe so that it just covers the top of the lobe, and surround the metal syringe with an ice cold chilling block. After one to two minutes, load the specimen syringe into the automated slicer, and align the fresh blade. Next, fill the buffer tank with ice cold PBS.Align the step motor drive with the specimen syringe, and remove the tape. Turn the switch to the fast forward position, until the step motor drive just touches the back of the plunger. Set the tissue thickness, continuous single slice, oscillation, and speed settings on the slicer.Then press start and use a thin spatula to collect each PCLS as it falls into the buffer tank, placing the slices into individual wells of a 24 well plate containing ice cold PBS. For static PCLS imaging, transfer the lung samples that contain the anatomical area of interest into individual three centimeter dishes containing one milliliter of the appropriate antibody cocktail of interest. After 30 to 60 minutes on ice with rocking and protected from light, rinse the slices two times with one milliliter of ice cold PBS.Next, add 50 microliters of PBS into one new, three centimeter round glass bottomed dish per sample, and quickly transfer the slices into each drop, gently manipulating the tissue pieces so they are spread flat. Then aspirate the PBS with a pipette, and place a drop of room temperature mounting medium onto the slide, followed by a glass cover slip. To image the tissue, place the dish onto a confocal microscope stage under a 20x objective, and use the tile tool to locate and mark the edges of the tissue in the convex hull setting to reduce the time of the tile scan.Then set the z-stack range, taking care that the set ranges are appropriate at multiple areas of the slice, particularly along the edges. For live cell imaging, after 30 minutes of incubation in the antibody cocktail of interest, rinse the slices two times with one milliliter of cold Leibovitz 10. Next, add 50 microliters of fresh Leibovitz 10 into one slot of a chambered cover glass per sample, followed by the immediate transfer of each PCLS to the medium, gently manipulating the tissues until they are spread flat.Then use a pipette to aspirate the medium from each chamber and anchor each PCLS with a platinum weight. Carefully pipette freshly prepared ice cold extracellular matrix onto each PCLS, taking care that the gel is placed on top of the lung slices, and that the slices do not float on top of the gel. After five minutes at 37 degrees Celsius, transfer the slide into a pre-warmed confocal microscope chamber set to 37 degrees Celsius under a 20x objective.Then, set the z-stack and tile ranges based on the desired interval between the frames, using the tile tool to denote the area of interest in the tissue, and the centered grid setting to center the sample in the field of view. CD11b high conventional dendritic cells localize in the lung parenchyma, whereas CD103 positive conventional dendritic cells reside primarily around the airways and the subpleural area. Co-staining for CD88 and CD172a, allows the distinction of CD172a negative, CD88 positive alveolar macrophages, from CD172a positive, CD88 positive interstitial macrophages, the latter of which are preferentially located in the parenchyma, and not the subepithelial area.Co-labeling for CD90.2, a well-known T-cell marker, and the TdTomato fluorescence protein, a transgene under the transcriptional control of a master transcription factor necessary for lymphagiogenesis, facilitates the identification of lymphatic structures in the lung. CD103 positive conventional dendritic cells are found in several anatomical areas, including the CD324 expressing airway epithelium, the lymphatics, and the subpleural area. Cell movement, and cell-to-cell interactions can be recorded using live cell imaging as just demonstrated.For example, in the representative experiment, 16 hours after oral ova-LPS installation, CD103 positive dendritic cells can be clearly observed interacting with adoptively transferred ova-specific T-cells over a four hour period. Once mastered, the tissue preparation and the slicing techniques can be completed in two and a half hours if they are performed properly. While attempting this procedure, it's important to remember to achieve a good agarose lung inflation, and to subsequently keep the delicate lung slices as flat as possible for imaging.Following this procedure, other methods, like treating PCLS with a acetylcholine or methacholine, can be performed to answer additional questions about calcium signaling and airway contractility under various disease conditions. After watching this video you should have a good understanding of how to generate precision cut lung slices for visualizing the localization, trafficking, and interactions of immune cells in the lung.

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Research

JoVE Journal

Immunology and Infection

16.6K ビュー.  National Institute of Environmental Health Sciences, NIH. The overall goal of this method is to use precision-cut lung slices, or PCLS, to visualize the localization and trafficking of various immune cell types in the lung. This method can help answer key questions in the immunology field, such as the localization and migration patterns of immune cells in the lung. The main advantage of this technique is that several interactions can be visualized in the live ex vivo tissue that maintains the three dimensional architecture of the lung.Though ...

ビデオ: Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...