For continuous labeling in vivo, BrdU (50 mg/ml) was administered to mice throughout a 7-day period via a subcutaneous miniosmotic pump (Alzet model 2001, Durect Corporation, Cupertino, CA). Alzet pumps were implanted in mice and removed after one week. Mice were sacrificed 4 weeks after removal of the Alzet pumps. Lungs were fixed overnight via tracheal instillation of fresh 4% paraformaldehyde and embedded in paraffin prior to sectioning. Label-retaining cells were identified by BrdU immunofluorescence. BrdU and CCSP double staining was performed and cells exhibiting a nucleus and attachment to basement membrane were counted. Bronchiolioles (BLs) were defined as intrapulmonary airways in which smooth muscle, but neither cartilage nor glands, could be seen. Terminal bronchioles (TBs) contained an intact bronchioalveolar duct junction (BADJ) and visible alveolar duct [17] . In TBs quantification of staining included all cells within 200 mm of the BADJ. A total 49 TBs and 26 BL structures were analyzed in lung sections of five mice. Figure 1 . Schema for purification of primary CCSP positive cells from mouse lung. A) Two month old FVB mice were euthanized by CO 2, lungs removed and lobes collected. After washing in PBS, lobes were minced on ice and incubated in a small cell culture dish with 3 mg/ml collagenase in PBS (total 5 ml) in a shaking platform for 1 hour at 37uC. The suspension was further disaggregated by trituration through a 19 gauge needle, with 5 ml of PBS, filtered through a 40 mm cell strainer and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, cells resuspended in red blood cell lysis buffer for 4 min re-plated into 10 cm culture dishes for recovery overnight (18 hrs). Surviving cells were adhering to the dish. After trypsinization and neutralization by 10% FBS media, cells were resuspended in PBS with 3% FBS, and stained with rabbit anti-CCSP antibody and FITC conjugated anti-rabbit secondary antibody. CCSP + and CCSP 2 cells were sorted with FACS Vantage SE cell sorter. B) Rabbit IgG was used as an isotype -matched negative control; CCSP + population sorted with FACS Vantage SE cell sorter from dissociated lung tissue was 25.37%. doi:10.1371/journal.pone.0043008.g001