Isolation and Characterization of Microliths

Synthetic hydroxyapatite particles (10 µM average size) were purchased from Fluidinova (article 501203, Moreira da Maia, Portugal). Mouse microliths were isolated from the lungs of 20- to 28-week-old male Npt2b^−/− mice after euthanasia by intratracheal instillation of 3 ml of dispase (50 caseinolytic units/ml, Corning, NY), submersion of the organ in 1 ml of dispase for 45 min at room temperature, and transfer to a culture dish containing water. The parenchymal lung tissue was gently teased from the bronchi, then homogenized. For some experiments, mouse microliths were isolated without dispase treatment. Human PAM microliths were isolated from the lung explant of a PAM infant undergoing lung transplant, by gently teasing the lung parenchyma apart with forceps in a culture dish containing water. Microliths were collected by centrifugation, and washed 5 times with cell culture grade water.