Sean Lauber:Broncho-alveolar Lavage

This procedure explains how to sacrifice the mouse (by bleeding/asphyxiation). Once the mouse is bled and the diaphragm is cut back the mouse is likely dead and the anasthetic nose tube can be removed. Before beginning the procedure be sure to prepare cannulas: 21 gauge needles fitted with 0.58 mm polyethylene tubing (Itramedic PE50 NO. 427411). These cannulas will be inserted into the trachea and then secured using short pieces of thread (be sure to have thread on hand). This cannula can then be fitted with a syringe and PBS can be used to fill the lung and then retracted for collection. This is the broncho-alveolar lavage.

This same procedure is required to isolate the lungs for formalin fixation.

1. Anesthetize mouse using isoflurane
2. Using a nose cone stuffed with isoflurane-soaked cotton, remove the mouse to the work bench and maintain anesthesia
3. Soak the mouse in EtOH and make an incision in the abdomen
4. Tear back the skin to expose the peritoneum
5. Cut back the peritoneum by cutting around the base of the ribs to expose the guts
6. Cut back the diaphragm, being sure not to cut the lungs, then cut the renal artery to bleed the mouse (note that here you can collect the blood using a 1 ml syringe)
7. Cut the ribs at an angle towards the neck of the animal on both sides (don't cut the lungs, use forceps to help push this tissue out of the way while you cut)
8. Pull the rib cage flap back to the head of the mouse and you should see the trachea
9. Remove any excess tissue around the trachea
10. Tie a piece of thread loosely around the trachea
11. Make an incision in the trachea small enough to permit a cannula. Be sure not to cut the trachea all the way through. If you do this there is little hope for continuing. Just "nick" the trachea.
12. Slide the cannula in
13. Secure the cannula using the piece of thread (make sure it is very tight - use the forceps to get a good grip)
14. Cut the trachea free from above the point of cannula insertion
15. Remove excess tissue by cutting with sharp scissors from below the trachea while pulling the lungs free
16. Instill 500 μL of sterile PBS (RT) into the lungs slowly
17. Massage the lungs to help drive the liquid out. Place the liquid on ice. This is your BAL and you should recover about 300 ul.
18. Repeat three more times with 500 ul PBS and collect this in a separate tube. This is to collect the bulk of the cells, keep it on ice.
19. When you go upstairs spin the BAL and cells down at 10 K RPM at 4*C for 2 minutes. Remove the supernatant and store the BAL in a new tube at -80*C.
20. Discard the supernatant from the other tube (containing about 1.5 ml)
21. Add about 100 ul of PBS and resuspend the pellet from the BAL tube, then combine this with the cells in the other tube. This is what you will use to count cells and perform cytocentrifugation.