Isolation and Purification of Alveolar Type II Cells

Mice were sacrificed by intraperitoneal injection of Euthasol, and lungs were perfused with 10 ml of sterile normal saline via the pulmonary artery. The airway was cannulated via tracheostomy with a 20-gauge metallic angiocatheter, and 3 ml of dispase (50 caseinolytic units/ml, Corning) was instilled, followed by 0.5 ml of 1% low-melt agarose (warmed to 45 °C). Lungs were rapidly cooled on ice for 2 min, submerged in 1 ml of dispase for 45 min at room temperature, and transferred to a culture dish containing deoxyribonuclease I (100 U/ml) (Worthington Biochemicals, Malvern, PA). The parenchymal lung tissue was gently teased from the bronchi, and homogenized. Cell suspensions were filtered, collected by centrifugation, and panned over prewashed 100-mm tissue culture plates coated with CD45 (#553076, monoclonal, 1:100) and CD16/32 (#553142, monoclonal, 1:100) antibodies (BD Biosciences, San Jose, CA). After incubation for 60 min at 37 °C in a 5% CO₂ atmosphere to promote adherence of contaminating macrophages and fibroblasts, the AT2 were gently decanted from the plate, collected by centrifugation, and counted. For the Npt2b^−/− animals, differential centrifugation was used to separate microliths from the cells. Cell viability determined with trypan blue staining was routinely >90%, and cell purity determined by SP-C staining ranged from 75 to 90%.