4.5. Collagen Content Assay

Total lung collagen content was measured using Total Collagen Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocol. In brief, every 10 mg of lung tissue was homogenized in 100 µL ddH₂O and mixed with equal volume 12M HCL. Then, samples were hydrolyzed at 120 °C for 3 h. After homogenization, 4 mg of activated charcoal was added into individual tubes and vortexed. After centrifuging at 10,000× g for 3 min, 15 µL supernatant of each vial was transferred to a 96-well plate and dried in a 70 °C oven. Reaction reagent was added into each well and absorbance was measured at 560 nm using a SpectraMax M5 multi-mode reader (Molecular Devices, San Jose, CA, USA)