Elastase solution

CC10-CreER; LSL-Sox2 mice and CC10-CreER; Rosa26-f-GFP mice were sacrificed 20 wk after tamoxifen injection. Lungs were perfused with 10 mL of PBS and lavaged four times with 1 mL of PBS and 0.2 μM EGTA. One milliliter of 4 U/mL Elastase solution (Worthington Biochemical) was instilled in the airways and incubated for 5 min at 37°C followed by three 0.5-mL instillations for 5 min each. Lung lobes were then minced and incubated with DNase I solution for 10 min at 37°C. Cells were passed through a 100-μm cell strainer, and red blood cells (RBCs) were lysed using RBC lysing buffer (eBioscience, Inc.). Isolated cells were resuspended at 1 × 10⁶ cells in 100 μL of Hanks’ balanced saline solution, 10 mM HEPES, and 2% fetal bovine serum. Propidium iodide (PI) was used for dead cell discrimination. GFP-positive cells were sorted and collected on FACS Vantage SE. RNA was then extracted using Qiagen protocols. The RNA was reverse-transcribed, and the cDNA was hybridized to Affymetrix 420 mouse microarray chips in the Duke Medicine microarray facility. The resulting CEL files were rma-normalized in Bioconductor in the R environment. Differential expression was carried out using the Limma package with multiple comparisons controlled by the method of Benjamini and Hochberg.