精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

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.