Whole Lung Agarose Inflation: A Method of Preparing Lungs for Ex vivo Live Imaging

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Lungs for Ex vivo Live Imaging

NOTE: Try to work as sterile and careful as possible to avoid unnecessary challenges of the immune cells within the lungs.

1. Inject the mouse intraperitoneal (i.p.) with a lethal overdose of an anesthetic    permitted by the animal protocol approved by IACUC, e.g., 1 ml of 2.5% Avertin. Wait for the mouse to stop breathing and be completely non-responsive to noxious stimuli (hind paw pinch).
   NOTE: Cervical dislocation and carbon dioxide euthanasia should be avoided as it can detrimentally affect lung cell viability.
2. Immobilize the mouse on a dissection board and sterilize the mouse with 70% ethanol.
3. Use surgical scissors to first make a transverse epigastric incision through the skin, followed by a similar incision through the peritoneum. Hold the dissection board in a vertical position and cut the descending aorta, so that blood pools down in the abdomen and not in the chest cavity.
4. Snip a small opening in the diaphragm to release vacuum. Cut along the 10th and 12th rib to excise the diaphragm and get visual access to the lungs.
5. Use surgical scissors to cut the skin up to the trachea over the ribcage but leave the ribcage intact. Separate the skin from the ribcage. Expose the trachea by removing the surrounding connective tissue, being careful not to damage the trachea itself (Figure 1A).
6. Snip a small opening approximately 1 mm in diameter in the exposed trachea parallel to the cartilaginous rings, as close to the larynx as possible (Figure 1B). Be careful not to cut completely through the trachea.
7. Take a 20 G needle and gently insert the needle 4-5 mm into the trachea without any counter force (Figure 1D). The end of the needle should be visible through the trachea (Figure 1C). Use forceps to stabilize the needle in the trachea. Alternatively, a suture may be tied around the trachea to hold the needle in place.
   NOTE: By inserting too deep, the carina may be traumatized or only one side of the lungs might be inflated.
8. Fill a syringe with 400 µl of 37 °C 2% low-melting-temperature agarose (taken directly from a constant temperature bath). Make sure the dissection board is standing up and slowly instill the warm agarose through the needle into the lungs, use ~400 µl to inflate the lungs.
   NOTE: Watch the lungs inflating inside the ribcage. Do not over-inflate the lung as it will rupture.
9. Once the lungs are inflated, filling ~⅔ of the rib cage, detach the syringe and keep the needle inside the trachea to prevent any agarose from leaking.
10. Pour approximately 50 ml of 20 °C PBS over the inflated lungs to allow the agarose inside the lungs to set and solidify. Slowly remove the needle and close the trachea with forceps to prevent any non-solidified agarose from leaking.
11. Expose the lungs by performing a sternotomy and subsequently excise the lungs. For excision of the lungs, hold on to the trachea while cutting through the trachea completely. Gently pull the trachea up, cut away the connective tissue and esophagus while pulling the lungs out of the chest cavity until the lungs is separated from the mouse (Figure 1E).
12. Immerse the lungs in warm RPMI-1640 to wash off excessive blood and gently separate the lobes by using scissors and forceps to cut the lobes’ main stem bronchus at hilum (Figure 1F).
13. Place the lobes, with the flat surface down to maximize imaging surface, in a well of a 24-well imaging plate (Figure 1G). Add 100 µl of 37 °C RPMI-1640 on top of the lobes. Place several 15 mm circular microscope cover slides on top of the lobes to prevent it from floating.
14. Pour warm PBS into the surrounding wells to prevent the RPMI-1640 media from evaporating. Insert the 24-well plate into the equilibrated climate chamber and maintain the lung lobes at 37 °C with air and 5% CO₂. Insert the climate chamber on the stage of the confocal microscope.
    NOTE: Other gas mixtures (e.g., 5% O₂, 5% CO₂ in N₂ to examine cell behavior under conditions of hypoxia/lower oxygen) could also be considered.