Rbc lysing buffer

At the indicated times post-infection, mice were anesthetized with isoflurane and administered 1 ug anti-CD45.2 antibody intravenously. After 5–10 minutes of in vivo incubation, mice were euthanized by CO₂ asphyxiation. Mouse lungs were excised and lightly homogenized in HEPES buffer containing Liberase Blendzyme 3 (70 μg/ml; Roche) and DNaseI (30 μg/ml; Sigma-Aldrich) using a gentleMacs dissociator (Miltenyi Biotec). The lungs were then incubated for 30 min at 37°C and then further homogenized a second time with the gentleMacs. The homogenates were filtered through a 70 μm cell strainer, pelleted for RBC lysis with RBC lysing buffer (Thermo), and resuspended in FACS buffer (PBS containing 2.5% FBS and 0.1% NaN₃).

Ten microliter of the appropriate conjugated anti-rat antibodies (CD4/FITC, CD11b/PE; Becton Dickinson, CA, USA) were added to 100 µL of peripheral blood from each animal and incubated at room temperature for 15 min in the dark. Erythrocytes were eliminated by adding 500 µL of RBC lysing buffer (ebioscience, USA) for 10 min. After incubation, leukocytes were washed twice in PBS. The washed cells were re-suspended in 0.3 mL of PBS. Cells were analyzed using a Beckman Coulter instrument equipped for four-color flow cytometry (Coulter, Coultronic, Margency, France), and up to 10000 events were obtained. Data were analyzed using the Kaluza analysis software (Beckman Coulter Inc., USA).

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.