Mak008

The collagen content in the heart was measured by determining the content of hydroxyproline [15 (link)]. The experiment was performed by using a colorimetric assay kit (MilliporeSigma, #MAK008), following the manufacturer’s recommendation with minor changes. Briefly, after homogenization, cardiac samples (about 20 mg of both ventricles) were weighed and resuspended in a volume of 50 μl H₂O per 10 mg of tissue. Acid hydrolysis was then done in 6 N HCl (vol/vol) for 180 min at 120 °C. The hydrolysate was cooled and centrifuged for 3 min at 10,000×g. The supernatant was collected, and 25 μl was added to a 96-well plate for the assay (in duplicate for each sample).
The drying out process was then performed in an oven at 60 °C overnight. The next day, hydroxyproline was first oxidized with 7% Chloramine T in the oxidation buffer; then, Ehrlich’s solution was added in the wells (1.4 M 4-dimethylaminobenzaldehyde in 20% perchloric acid and 67% isopropanol) and the plate was incubated at 60 °C for 90 min. After cooling, absorbance was read at 560 nm. The hydroxyproline content was calculated using a standard curve of high-purity hydroxyproline and finally reported per milligram of the heart weight.

A hydroxyproline assay kit (MAK008, Sigma, St. Louis, MO, USA) was used to detect the content of hydroxyproline in mouse right lung [26 (link)]. Briefly, the fresh lung tissue was homogenized in water (100 µL water of every 10 mg lung tissue), followed by hydrolyzing the samples after adding equivalent 12 N HCl for 3 h at 120 °C. Next, the samples were centrifuged (12,000 rpm, 4 °C, 3 min; Beckman Microfuge 20R) and extracted 10 µL supernatant of every sample, the samples were transferred to a 96-well plate and evaporated at 56 ℃. The samples in the plate were incubated for 5 min at 20 ℃ after added 100 µL chloramine T reagent into each well. Afterward, the samples were incubated for 90 min at 56 °C after mixed with 100 µL p-dimethylaminobenzaldehyde reagent. Absorbance of samples was measured by BioTek ELx800 plate reader at λ = 560 nm (Winooski, VT, USA). The hydroxyproline content of samples was calculated by a standard sample and presented as µg hydroxyproline per right lung.

Diaphragm muscles were excised, carefully cleaned to remove attached tendons, weighed, and snap frozen in liquid nitrogen. Collagen content was measured using a hydroxyproline assay kit (MAK008; Sigma-Aldrich). Briefly, diaphragm muscles were homogenized in water and hydrolyzed overnight in 6 M hydrochloric acid at 110°C. 10 µl of homogenate was then transferred to a 96-well plate and incubated at 60°C to dry the sample. Dried samples were resuspended in chloramine T/oxidation buffer mixture, incubated at room temperature for 5 min, mixed with DMAB-perchloric acid/isopropanol reagent, and then incubated for 90 min at 60°C. Sample absorbance was measured at 560 nm using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). Absorbances (ODs) obtained for each sample were referred against a standard curve to quantify the amount of hydroxyproline, which was then normalized to milligrams of tissue wet-weight.

Native (n = 3) and decellularized (n = 3) samples of the pulmonary wall, leaflet, and myocardium (3–5 mg each) were used for this quantification. Hydroxyproline extraction was performed using a hydroxyproline assay kit (MAK008, Sigma-Aldrich) [30 (link)]. Absorbance was measured at 560 nm with a spectrophotometer reader (SPARK).

Picro‐Sirius red staining was performed using a commercially available kit (ab150681; Abcam) according to the manufacturer's protocol. Tissue collagen accumulation was quantitatively assessed by a hydroxyproline accumulation assay (MAK008; Sigma‐Aldrich) according to the manufacturer's instructions.