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;ve mice and in mice treated with 100 &#181;g ovalbumin (OVA) and 0.1 &#181;g lipopolysaccharide (LPS), using phosphate buffered saline (PBS) as a vehicle in a total volume of 50 &#181;L. To oropharyngeally instill OVA/LPS into the airways, animals were anesthetized with isoflurane inhalation and vertically suspended by their teeth with a rubber band. The tongue was gently grasped with forceps and held to one side to prevent swallowing, and 50 &#181;L of the OVA/LPS solution deposited at the back of the oral cavity as previously described17.</li>
	<li>Euthanize the mouse with intraperitoneal injection of sodium pentobarbital (100 mg/kg), and pin the animal to a polystyrene base.</li>
	<li>Open the abdominal cavity with scissors by cutting the skin and peritoneum from the middle of the abdomen up to the jaw. Pull the intestines aside with forceps or the dull edge of the scissors, and snip the inferior vena cava to drain blood away from lungs. Puncture the diaphragm with the sharp tip of the scissors to allow expansion of the rib cage, being careful not to cut the lungs.</li>
	<li>Clear salivary glands and other tissue away from the trachea by grabbing the tissue and manually pulling it away from the underlying trachea with forceps. Make a small incision in the trachea on the anterior side of the thickest band of cartilage using fine forceps or scissors, being careful not to cut all the way though the trachea. The incision will be just large enough to allow a 20 G needle to pass through.</li>
	<li>Slide a 1.5 inch, 20 G needle onto a section of polyethylene tubing, leaving approximately 1 cm of tubing beyond the end of the needle. Cut the tubing at an angle of approximately 45&#176; to bevel the end, attach the needle to a 1 mL syringe, and load the syringe by drawing 0.8 mL of warm (40 &#176;C) 2% low melting point agarose up through the needle.</li>
	<li>Place the end of the tubing into the incision of the trachea and slowly inject the agarose into the lungs. Without moving the needle or syringe, tape the syringe to the polystyrene base to ensure the needle remains in the trachea. 
	NOTE: After injection, the lungs will be larger and inflated within the chest. The right lobes expand first, then the left lobe. If only one lobe expands, the needle has been inserted too deeply (past the bronchus), and it will be necessary to pull it out slightly before proceeding. This part of the procedure needs to be done relatively quickly, before the agarose starts to solidify.</li>
	<li>Place the mouse, with the base and the syringe still inserted in the trachea, in a cold room or refrigerator for at least 10 min. If necessary, the animal can be kept there for up to several hours, even if proceeding to image cell migration by video microscopy.</li>
	<li>Once the mouse is cold, carefully excise the agarose-inflated lungs using scissors, and place them in a 3 cm dish with ice cold PBS. Keep on ice.</li>
	<li>Choose the desired lobe for tissue slicing (e.g., the right superior lobe). Make sure the interior part of the lung (where it connects to trachea) is face down in the dish. 
	NOTE: The lobe used and its orientation on the plunger will determine the size of vessels and airways that will be visible. The orientation described here is ideal for visualization of large airways, and parenchyma. Mice have one large left lobe and four smaller lobes on the right. For PCLS imaging following introduction of allergens into the lungs via oropharyngeal or intranasal aspiration, the right superior lobe is typically sectioned, in part because it is a convenient size, but also because inhaled agents disperse uniformly within it. However, other lobes, especially the left lobe, can also be studied. When comparing mouse strains or treatments, it is important to compare the same lobe.</li>
</ol>
2. Lung Slicing
NOTE: Make slices using an automated slicer, metal cooling block, plunger and metal syringe, according to the manufacturer&#39;s instructions.
<ol>
	<li>Put a small drop of all-purpose, no-run gel superglue onto the plunger and spread the glue in a circular motion, taking care to not let the glue touch the sides of the metal syringe. If the glue touches the sides of the syringe, it will glue the syringe and plunger together.</li>
	<li>Immediately after covering the plunger with glue, gently grasp the excised lobe with forceps with the trachea side down, dab it on a tissue to remove excess liquid, and carefully place it on top of the plunger. Trim off any extra tissue extending beyond the edge of the plunger.</li>
	<li>Move the plunger down so that the sides of the metal syringe move up and over the tissue, creating a well with the lung glued at the bottom, no more than several centimeters deep. Tape around the bottom of the metal syringe to hold this position in place.</li>
	<li>Carefully pour 2% low melt agarose at 40 &#176;C into the well so it just covers the top of the lobe.</li>
	<li>Surround the metal syringe with the ice-cold chilling block and cool the lobe and agarose for 1 - 2 min. 
	NOTE: A shorter time cooling of the agarose surrounding the tissue could lead to disintegration of the agarose during slicing, which might result in an unevenly cut PCLS.</li>
	<li>Load the specimen syringe into the automated slicer and fill the buffer tank with ice cold PBS. Align the step motor drive with the specimen syringe and remove the piece of tape from the bottom. Turn the switch to the fast forward (FF) position until the step motor drive just touches the back of the plunger.</li>
	<li>Align the fresh blade with the specimen syringe. Set tissue thickness, continuous/single slice, oscillation and speed in the slicer. For example, tissue thickness: 150 &#181;m, continuous cutting (cont.), oscillation: 9, and speed: 3 - 4. Press start. The above settings need to be adjusted to the specific automated slicer specifications.</li>
	<li>Using a thin spatula or paintbrush, collect the PCLS one at a time as they fall into the buffer tank and place them in a 24-well plate containing ice cold PBS. 
	NOTE: It is important to keep the slices in order to maintain consistency between experiments. Although every lung is different depending on age, gender and weight, the 10th slice, for example, generally yields similarly sized airways.</li>
</ol>
3. Antibody Staining
<ol>
	<li>Design a panel of fluorescently tagged antibodies based on the cell type of interest, taking into account potential fluorescent spectral overlap and the detection capabilities of the microscope. 
	NOTE: As an example, a panel for DC subset detection is as follows: CD11c/alpha X integrin - BrilliantViolet (BV) 605 (Excitation: 405 nm, Peak emission: 605 nm), CD324/E-cadherin - Alexa 488 (AF488) (Excitation: 488 nm, Peak emission: 519 nm), CD88/C5Ra1 - Phycoerythrin (PE) (Excitation: 561 nm, Peak emission: 578 nm), CD103/alpha E integrin - Allophycocyanin (APC) (Excitation: 633 nm, Peak emission: 660 nm). Fluorescence spectral viewer tools (see Materials List) are useful to design panels.</li>
	<li>Make an antibody cocktail containing the desired antibodies.
	<ol>
		<li>For static PCLS imaging, add 800 &#181;L staining buffer, 100 &#181;L Fc blocker, 50 &#181;L normal mouse serum, and 50 &#181;L normal rat serum to a 1.5 mL microcentrifugation tube for a total volume of 1 mL. For live cell imaging, use 800 &#181;L Leibovitz&#39;s medium (1x) containing 10% FBS (Leibovitz-10) instead of staining buffer.</li>
		<li>Add antibodies to adjust a desired final concentration of each antibody. Transfer the antibody solution to a 3 cm dish, and keep in the dark until ready to use. 
		NOTE: The final concentrations need to be optimized for each individual antibody (usually 1 - 5 &#181;g/mL).</li>
	</ol>
	</li>
	<li>Choose a PCLS with an anatomical area of interest, such as large airways or periphery of the lung, and place in the 3-cm dish containing 1 mL antibody solution.</li>
	<li>Stain PCLS in the dark, on ice, rocking slowly for 30 or 60 min.</li>
	<li>For static PCLS imaging, proceed to the section 4. For live cell imaging, proceed to section 5.</li>
</ol>
4. Static Imaging of PCLS
<ol>
	<li>After 60 min incubation of PCLS with antibodies, remove the antibody solution from the dish and rinse the slices twice with 1 mL ice cold PBS.</li>
	<li>Pipette 50 &#181;L PBS onto a 3 cm round glass-bottom dish. Using a spatula or paintbrush, transfer the stained PCLS to the drop, gently manipulating the slice until it is flat and spread out. Remove the PBS with a pipette. The slice should lie as flat as possible. The above procedures need to be performed quickly to avoid photobleaching.</li>
	<li>Place 1 drop of room temperature mounting medium onto the slice and gently drop a&#160;glass coverslip into the well of the plate. Keep PCLS in the dark at 4 &#176;C for up to an hour before imaging.</li>
	<li>When viewing under a confocal microscope, use a 20X objective (see Materials List). Use the &#34;tile&#34; tool to locate and mark the edges of the tissue in the &#34;convex hull&#34; setting. This will help reduce the time of the tile scan.</li>
	<li>While setting the z-stack range, verify at multiple areas of the slice, especially along the edges, that the set ranges are appropriate. 
	NOTE: The z-range will likely need to be greater than the thickness of the tissue itself because it is very difficult to get the tissue to lay completely flat. For example, with a 150 &#181;m thick PCLS, the z-stack range will likely need to be set to 200 - 250 &#181;m to accommodate bumps and curves in the slice. Depending on the z-stack range and the size of the tissue, each full lung scan will take between 7 - 12 h with a 20X objective. Each lung is unique and the settings must be adjusted for every experiment.</li>
</ol>
5. Live Cell Imaging of PCLS
<ol>
	<li>After 30 min incubation of PCLS with antibodies, remove the antibody solution from the dish and rinse the slices twice with 1 mL cold Leibovitz-10.</li>
	<li>Pipette fresh 50 &#181;L Leibovitz-10 into one slot of a chambered coverglass (8 slots). Use a spatula to transfer the PCLS to the medium, gently manipulating it until the slice is flat and spread out. The above procedures need to be performed quickly to avoid photobleaching.</li>
	<li>Remove the 50 &#181;L medium using a pipette. The slice is to be lying as flat as possible. 
	NOTE: It is acceptable if the edges of the slice are flipped up vertically against the wall of the chamber. Furthermore, if available, a metal platinum weight can be used to anchor the slice before proceeding to the next step. See the attached Materials List for one example of platinum wire that can be cut and bent into small weights.</li>
	<li>In a 4 &#176;C cold room, mix 100 &#181;L of growth factor-reduced, phenol red-free extracellular matrix with 100 &#181;L Leibovitz-10. Use precooled pipette tips for this, or the extracellular matrix will solidify in the tip. 
	NOTE: This ratio of matrix and medium (1:1) creates a gel dense enough to hold the PCLS down in tissue-culture conditions.</li>
	<li>Gently pipette the matrix to mix, and carefully pipette on top of the PCLS, making sure that the gel goes on top of the lung slice, and that the slice does not float up on top of the gel. The gel should work as a weight, anchoring the PCLS down onto the bottom of the plate.</li>
	<li>Carefully transfer the chambered coverglass to a 37 &#176;C incubator and let the matrix solidify for ~5 min.</li>
	<li>Transfer the entire chambered coverglass onto the pre-warmed stage of a confocal microscope. Maintain the chamber at 37 &#176;C throughout imaging. CO2 supply is not required during the cell incubation when using Leibovitz&#39;s medium.</li>
	<li>Set the z-stack range and tile range based on the desired interval between frames. When viewing under a confocal microscope, use a 20X objective (see Materials List). Use the &#34;tile&#34; tool to denote the area of interest in the tissue, using the &#34;centered grid&#34; setting (e.g., a 2x2 centered grid).</li>
	<li>While setting the z-stack range, verify at multiple areas of the slice that the set ranges are appropriate. 
	NOTE: The z-range will likely equal the thickness of the tissue. Each lung is unique and the settings must be adjusted for every experiment. 2x2 tiles and 10 z-stacks will generally result in ~1 frame/2 min. Ensure the auto-focus function is activated before starting the experiment to minimize x-y and z-drift during the imaging period.</li>
</ol>

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The authors have no conflict of interest to declare.

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 ...

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

Research

JoVE Journal

Immunology and Infection

16.8K Views.  National Institute of Environmental Health Sciences, NIH. 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.

Video: 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 ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

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 ...

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.

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 ...

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

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Rozdziały w tym wideo

Isolation of Mouse Lung Dendritic Cells

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

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

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

Pseudomonas aeruginosa Induced Lung Injury Model

Influenza Virus Propagation in Embryonated Chicken Eggs

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

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

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

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-α