Fibrinolytic niche is requested for alveolar type 2 cell-mediated alveologenesis and injury repair

Ali Gibran; Runzhen Zhao; Mo Zhang; Krishan G. Jain; Jianjun Chang; Satoshi Komatsu; Xiaohui Fang; Beiyun Zhou; Jiurong Liang; Dianhua Jiang; Mistuo Ikebe; Michael A Matthay; Hong-Long Ji

Mouse AT2 cells were isolated from wt, Plau -/-, and Sepine1 Tg strains of C57BL/6 animals (Jackson Laboratory, USA) as previously reported with modifications1. Briefly, mice were euthanized and exsanguinated, followed by perfusing lungs with 10 -20 mL DPBS until pink lungs turned to white. The trachea was cannulated with a 20G catheter to instill 1.5 -2.0 mL dispase followed by 0.5 mL of 1% low melting point agarose. The lungs were dissected and incubated in 50 U/mL dispase solution for 45 min at room temperature. The lungs were gently teased in DMEM/F-12 + 0.01% DNase I and incubated for 10 min at room temperature. Cells were passed through a serial filtration (100, 40, 30, and 10 µm cell strainers) and centrifuged at 300 × g for 10 min at 4 o C. Cells were resuspended in 10 mL medium (DMEM/F-12 + 10% FBS + P/S) supplemented with biotinylated antibodies, rat anti-mouse CD16/32 (0.65 µg/million cells), rat anti-mouse CD45 (1.5 µg/million cells), and rat anti-mouse Ter119 (5 µg) and incubated for 30 min at 37 o C on an incubator shaker at 60 rpm. Resuspended cells were then incubated with pre-washed streptavidin-coated magnetic particles for 30 min at room temperature to remove undesired cells. Selected cells were then resuspended in 10 mL medium (DMEM/F-12 + 10% FBS + P/S) and incubated for 30 min at 37 o C in a sterile Petri dish to allow residual fibroblasts to adhere to the bottom of the dish.