Hydroxyproline hyp content assay kit

3 × 10⁵ dLSECs or dHUVECs were cultured in the bottom well in 12 mm transwell plate (Corning, 3462) and dried mouse decellularized matrix or lyophilized human cirrhotic liver tissues were placed in the insert well with 3.0 µm pore polyethylene terephthalate (PET) membrane. The samples were incubated at 37 °C for 90 h. The HYP content of supernatant in insert well (H1), of supernatant in bottom well (H2), and of remaining samples in insert well (H3) were detected by Hydroxyproline (HYP) Content Assay Kit (Solarbio, BC0255) according to the manufacturer's instructions. The proportion of sample degradation was determined according to the Equation (2)
$$\mathrm{Sample}\mathrm{degradation}=\frac{\mathrm{H}1+\mathrm{H}2}{\mathrm{H}1+\mathrm{H}2+\mathrm{H}3}\ast 100\%$$ The HYP content of liver tissue samples were also characterized Hydroxyproline (HYP) Content Assay Kit (Solarbio, BC0255) according to the manufacturer's instructions.

Collagen levels were measured by detecting hydroxyproline using the Hydroxyproline (HYP) Content Assay Kit (BC0250, Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, synchronized populations of worms were cultivated at 20 °C until they reached the late L4 stage, and then shifted to 15 and 20 °C to determine collagen contents. Forty to seventy milligrams of 1-day-old and 8-day-old worms were collected and washed three times with M9 buffer. Worms were homogenized in liquid nitrogen. The resulting powders were pre-digested in 0.5 ml of 6 M HCl in microwave at 110 °C for 2–6 h. Once the solution was clear, 6 M HCl was added to achieve a final volume of 0.5 ml. Collagen contents were determined using the kit according to the manufacturer’s instructions. The collagen contents were expressed as micrograms of collagen per milligram of worm wet weight.

Collagen assay was conducted as described previously [30 (link)]. Wild-type worms from the control and HLB01 (200 μg/mL) groups were cultured according to the lifespan assay. At day 10, worms were collected and washed by M9 for three times. Thereafter, collagen levels were measured by detecting hydroxyproline using the Hydroxyproline (HYP) Content Assay Kit (Solarbio, BC0250) according to the manufacturer's instructions. Three replicate experiments were conducted. An unpaired t-test was used to calculate the P values, and error bars represented SEM.