MassIVE MSV000083163

Description

Primary airway epithelial cells were isolated from WT and Club LRP1-/- mice as in Oreffo et al (Isolation of Clara cells from the mouse lung. Environmental health perspectives. 1990;85:51-64). Briefly, mice were anesthetized with isofluorane and their trachea was cannulated. Abdominal cavity was opened, and heart was perfused with PBS until the lungs and liver were free of blood. The lungs were then lavaged through the tracheal cannula with 1 mL saline, followed by protease solution (0.25% crystalline trypsin in 133 mM NaCl, 5.2 mM KCl, 1.89 mM CaCl2, 1.29 mM MgSO4, 2.59 mM phosphate buffer, pH 7.4, 10.3 mM Hepes buffer , pH 7.4, glucose 1 mg/ml), and then infused with protease solution according to the standard lung perfusion guidelines by the American Thoracic Society (61). Lungs were maintained filled with protease solution at 37 C for 15 min. Then, lungs and airways were extracted from the chest cavity and dissected in protease solution supplemented with fetal bovine serum. Trachea and major bronchi were removed and the parenchyma was diced in small pieces, placed in solution B (5.2 mM KCl, 2.59 mM phosphate buffer, pH 7.4, 10.3 mM Hepes buffer, Ph 7.4, glucose 1 mg/ml) and manually shaken to release the digested cells. The cell suspension was then filtered through gauze and 100 and 40 um mesh. The primary cell digest was centrifuged twice at 32 x g for 6 min at 10 C in 4 mL solution B with 250 ug/mL DNAse and the final pellet collected. For proteomic analysis. Airway epithelial cells collected as above were submitted to Bioproximity LLC (Chantilly, VA, USA). For proteome profiling, cells were trypsinized and equal amounts of protein per sample were used for UPLC-MS/MS for identification of protein IDs. [doi:10.25345/C5H02X] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: lung ; club cell ; COPD ; lipoprotein receptor ; LRP1 ; oxidation

Contact