APPENDIX II: Harvesting Bronchoalveolar Macrophages (BAM) via Lung Lavage

1. Rat is anesthetized by I.P. injection of sodium pentobarbitol (65 mg/kg B.W.)

2. Ventral side is swabbed with 70% ethanol and the skin and hair removed from the abdomen to above the throat

3. The muscle tissue is removed from the neck exposing the trachea

4. The trachea is cannulated with a blunt 16 gauge needle and fixed with surgical thread

5. The rat is then sacrificed by exanguination of the abdominal aorta

6. Following attachment of a three-way valve and a 0.45 micron syringe filter, warm (37°C) PBS is instilled into the lungs (35 mL/kg B.W.)

7. The thoracic cage is massaged, the valve is switched and the lavage fluid is collected into a second syringe

8. PBS is repeatedly instilled and removed until a total of 40 mL has been collected

9. The lavage fluid is then centrifuged (1500 rpm; 10 min) to pellet the cells

10. The cells are then resuspended in 20 mL of cold PBS and counted using a Coulter Counter

11. The cell suspension is spun a second time and resuspended in culture medium to the desired cell concentration