Hydroxyproline assay

10mg of normal and decellularized 3% Triton and 0.1% Triton liver samples were hydrolyzed with 6N HCL at 120C for 3h. Hydroxyproline content was then measured using a hydroxyproline assay (Sigma, St-Louis, MO) according to the manufacturer’s instructions. The collagen content was calculated indirectly through measurement of Hydroxyproline content according to Jamall et al. (1981) (link). The results were normalized to the wet weight of normal liver.

Hydroxyproline content was measured using a Hydroxyproline assay (Sigma-Aldrich) according to the manufacturer’s protocol and expressed as micrograms per lung.

Before digestion of hydrogels, each construct was weighed, and excess water was removed by using weighing paper. PBE buffer was prepared by weighing out 7.1 g Na₂HPO₄ and 1.86 g Na₂EDTA in 500 mL of ddH₂O and pH corrected to 6.5. The solution was sterile-filtered with a 0.22 um filter before use. Constructs were digested and homogenized in a papainase solution composed of 1.67 mg per mL of L-Cysteine (Sigma-Aldrich), PBE buffer, and 0.5% v/v papain (Sigma-Aldrich). Samples were incubated in this solution for 15–18 hours at 60°C. Samples were then frozen at −20°C until analysis. The cell density in each construct was quantified using the Quant-iT™ PicoGreen® assay following manufacturer’s instructions.
To determine collagen content, samples were first homogenized in ultra-pure water and then equal volume of 10N HCl was added to a glass PTFE-lined vial. Samples were hydrolyzed for 3 hours at 120°C. Once cooled, 5 mg of activated charcoal was added to samples. The supernatant was recovered by centrifugation. Samples were stored at −20°C until analysis. Using the manufacturer’s instruction for the hydroxyproline assay (Sigma-Aldrich), samples were mixed with Chloramine T/Oxidation buffer and diluted DMAB reagent. Samples were incubated in these two components for 90 minutes at 60°C before absorbance was recorded at 560 nm. Data was normalized to wet weight of hydrogel.