The heart, lungs and trachea were removed en bloc from mice following euthanasia by carbon dioxide inhalation. Lungs were separated and lobes minced on ice and incubated with collagenase type I (Invitrogen, Grand Island, NY) at 3 mg/ml in PBS in a volume of 2 ml per lung for 1 hour at 37uC with continuous agitation in an incubator. The suspension was further disaggregated by trituration through a 19 gauge needle (Sherwood Medical Co, St. Louis, MO), diluted in PBS. The crude cell suspension was filtered through a 40 mm cell strainer (BD Biosciences, Sparks, MD) and centrifuged at 700 rpm for 5 min. After discarding supernatant, cells were resuspended in 2 ml of red blood cell lysis buffer (eBioscience, San Diego, CA) for 4 min. Neutralization was performed with 10 ml of Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% FBS (Invitrogen) and cells were centrifuged at 700 rpm for 5 min. Cells were resuspended in DMEM/10% FBS with 20 ng/ml gentamycin/0.5 ng/ml amphotericin B (Cascade Biologicas tm , Portland, Oregon), plated in 100 mm dishes and placed to recover in an incubator at 37uC and 5% CO 2 for 18 hours ( Figure 1A ).