Protease inhibitor 1x

Media from 100mm cell culture plates with confluent primary lung fibroblasts from all three Oim genotypes were collected every other day in complete DMEM/F12 plus ascorbic acid (100ug/ml) to stimulate collagen production. Samples were then stored at −80°C. Media were then thawed on ice, transferred to a 15 ml Falcon, and centrifuged at 0.5G for 5 min to eliminate potential dead cells. Then protease inhibitor (1X) (Thermo Fisher cat # 1860932) and 25% V/V of saturated ammonium sulfate was added and put in rotation at 4 °C O/N. Next day, the samples were centrifuged at 25,000 at 4 °C for 90 minutes. The resulting pellets were re-suspended in 1.5 ml of pepsin (0.1mg/ml in 0.5 M acetic acid) and placed in rotation at 4 °C O/N. The next day NaCl dissolved in acetic acid was added to a final concentration of 0.9 M and the samples were put in rotation at 4 °C O/N to precipitate type I collagen. Samples were then spun at 14,000 RPM for 1 hour at 4 °C, the resulting pellets were then washed with 70% ETOH, and centrifuged again at the same speed and temperature for 30 min. After the pellets were dried, they were re-suspended in 0.1 M acetic acid. Collagen preparations were then loaded on a 6% gel-urea-SDS in non-reducing conditions, run for 2h and 30 min, and stained for 1 h with Coomassie blue. After the gel was destained, images were taken using a LICOR Odyssey CXL.