Mouse lung tissue dissociation and flow cytometry

Lung tissues were dissociated with a collagenase/dispase solution as previously described³¹. Briefly, after lungs were cleared by perfusion with cold PBS, 2 ml of dispase (BD Biosciences, 50Uml) was instilled into the lungs through the trachea until the lungs inflated. Each lobe was dissected and minced into small pieces in a conical tube containing 3 ml of PBS, 60 μl of collagenase/dispase (Roche), and 7.5 μl of 1% DNase I (Sigma) followed by rotating incubation for 45 min at 37°C. The cells were then filtered sequentially through 100- and 40-μm strainers and centrifuged at 1500 rpm for 5 min at 4°C. The cell pellet was resuspended in 1ml of RBC lysis buffer (ACK buffer, 0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA) and lysed for 2 min at room temperature. 8 ml basic F12 media (GIBCO) was added and 500μl of FBS (Hyclone) was slowly added in the bottom of tube. Cells were centrifuged at 1500 rpm for 5 min at 4°C. The cell pellet was resuspended in PF10 buffer (PBS with 10% FBS) for further staining. The antibodies used were as follows: CD45 (30-F11)-APC or -APC-Cy7 (BD Biosciences), CD31 (MEC13.3)-APC (BD Biosciences), EpCAM (G8.8)-PE-Cy7 or FITC (BioLegend), and CD24 (M1/69)-APC (eBioscience), MHC-II (I-A/I-E, M5)-FITC or -APC-Cy7 (eBioscience), and CD309/Kdr/Flk-1 (7D4-6)-APC (BioLegend). 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma) was used to eliminate dead cells. Data were acquired on LSRII analyser (BD Bioscience) and then analysed with FlowJo software (Tree Star).