Pe cy7 rat anti mouse cd31

Single cell suspensions of whole lungs were prepared as described previously [83 (link)]. For FACS, single cell suspensions were incubated with eBioscience Fixable Viability Dye eFluor™ 780 (Invitrogen) and the following antibodies (0.6 μg per 10⁷ cells): PerCP-Cy5.5 Rat Anti-Mouse CD45 (BD Pharmingen, 550994), PE-Cy7 Rat Anti-Mouse CD31 (BD Pharmingen, 561410), and Brilliant Violet 421 anti-mouse CD326 (Ep-CAM) (BioLegend, 118225) on ice for 45 min, and then subjected to FACS-sorting using FACS Aria II (BD Biosciences) at the Moody Foundation Flow Cytometry Core Facility at the Children’s Research Institute at UTSW. Flow cytometry data were analyzed with FlowJo v10.

For cell culture and fluorescence in situ hybridization (FISH), control, G4 Tert^−/− and pdgfα mice were sacrificed in homeostasis. For RNA‐seq, control and G4 Tert^−/− mice were sacrificed on post‐PNX Days 0 and 14. Then the digestive enzyme solution containing neutral protease (5 U/ml, LS02111, Worthington) and DNase I (0.33 U/ml, 10104159001, Roche) were injected into the trachea. After incubating for 45 min in digestive enzymes solution, the lung was cut into small pieces and vortexed on low power for 10 min. The cell mixture was filtered through 100 and 40‐μm strainers and incubated in red blood cell lysis buffer for 5 min. The cells were stained with antibodies as follows: PE‐Cy™7 rat anti‐mouse CD31 (1:400, B&D, 561410) and PE‐Cy™7 rat anti‐mouse CD45 (1:400, B&D, 552848). AT2 cells and pdgfα⁺ stromal cells were sorted for selecting GFP⁺CD31⁻CD45⁻ cells using the single‐cell module on the BD fluorescence‐activated cell sorting (FACS) Aria fusion I appliance.