Fastin elastin assay

Parts of the decellularized scaffold were fixed in formalin, embedded in paraffin, then sectioned at five microns, and transferred to glass slides for further histochemical stainings. Efficiency of the decellularization process was assessed qualitatively by hematoxylin and eosin (HE) and Feulgen staining. The quantity and length of remaining DNA was assessed using a blood and tissue kit (Qiagen) for DNA extraction. The extracted DNA was analyzed by agarose gel electrophoresis and quantified by incorporation of pico-green. Briefly, total DNA was isolated by phenol/chloroform extraction. The amount of remaining DNA was quantified by PicoGreen (Invitrogen) according to the manufacturer’s protocols. Qualitative histological analyses were performed to visualize the structural preservation of the scaffold and to determine the presence, distribution, and relative quantity of constitutive components of the tissue. Briefly, HE, Masson’s Trichrome, Elastica van Gieson staining were used to examine overall tissue structure, maintenance of collagen, glycosaminoglycan and elastin content organization. Quantification of the structural protein content was conducted using a sircol collagen assay and a fastin elastin assay (both Biocolor) according to the manufacturer’s protocols. All results were compared with native tissues as a control.

Elastin and soluble collagen levels were investigated in control VSMCs, calcifying VSMCs and osteoblasts after 7 and 14 days of culture. To measure elastin, cell layers were treated with 0.25% trypsin for 10 min to detach the cells. The resulting suspension was spun at 2,500 rpm for 5 min to pellet the cells and associated proteins. Cell-associated elastin was converted to water soluble α-elastin using 0.25 M oxalic acid and heating for 1 h at 100 °C. Elastin levels were measured using the Fastin™ elastin assay (Biocolor Ltd, County Antrim, UK) according to the manufacturer's instructions.
To measure soluble collagen, cells were transferred to αMEM containing 5% FCS and the lysyl oxidase inhibitor β-aminoproponitrile (50 μg/ml) for the final 24 h of culture. The concentration of collagen accumulated in the tissue culture medium was assayed using a Sirius red dye-based kit (Sircol soluble collagen assay, Biocolor Ltd) according to the manufacturer's instructions. For both assays, samples were normalised to total cell protein using the Bradford assay (Sigma-Aldrich, Poole, UK).

The elastin content of native and decellularized tissue was quantified using the FASTIN elastin assay (Biocolor, UK) according to the manufacturer’s instructions. Briefly, the samples were homogenized, and elastin was solubilized in 0.25 M oxalic acid. Two consecutive incubations were performed at 95°C to ensure complete extraction of elastin. Extracts were incubated with 5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonate (TPPS) dye, and absorbance was determined at 555 nm spectrophotometrically (Tecan Infinity, US). Elastin concentrations from a standard curve were used to calculate the elastin content of the tissue. For all quantifications at least three biological and technical replicates were obtained.

Elastin and glycosaminoglycan were quantified as previously described^{26 (link)}.
The elastin content of native and decellularized tissues was quantified using the FASTIN elastin assay (Biocolor) according to the manufacturer’s instructions. Briefly, the samples were homogenized and elastin was solubilized in 0.25 M oxalic acid. Two consecutive incubations were performed at 95 °C to ensure complete extraction of elastin. Extracts were incubated with 5,10,15,20-tetraphenyl-21H,23H-porphine tetrasulfonate (TPPS) dye and absorbance was determined at 555 nm spectrophotometrically (Tecan Infinity). Elastin concentrations from a standard curve were used to calculate the elastin content of the tissue.
The Glycosaminoglycan (sGAG) content of native and decellularized tissues was quantified using the Blyscan GAG Assay Kit (Biocolor). Briefly, the tissues were digested with papain at 65 °C for 18 h and aliquots of each sample were mixed with 1,9-dimethyl-methylene blue dye and reagents from the GAG assay kit. The absorbance at 656 nm was measured spectrophotometrically (Tecan Infinity) and compared to standards made from bovine tracheal chondroitin-4-sulfate to determine the sGAG content.

After myograph experiments, collagen and elastin content within the detrusor was determined. Mucosa was carefully removed, and detrusor was weighed. Then, 3 mg of detrusor was collected, and collagen was measured using Sircol™ Collagen Assay from Biocolor (Carrickfergus, UK) following the manufacturer's instructions. Briefly, tissue was minced using a fine scissor and digested with 0.1 mg/ml pepsin overnight at 4°C. This solution was centrifuged at 12,000 g for 10 min, and supernatant was carefully transferred to a clean tube and treated with an acid-neutralizing regent included in the assay kit. The collagen content was then determined using the Sircol Dye Reagent (Gray et al., 2008 (link); Toosi et al., 2008 (link)).
Similarly, elastin was measured using Fastin™ Elastin Assay (Biocolor) following the manufacturer's instructions. Elastin was extracted with 0.25 M oxalic acid at 100°C for 2 h and then precipitated using an Elastin Precipitating Reagent included in the assay kit. The elastin content was measured using the Elastin Dye Reagent (Gray et al., 2008 (link); Toosi et al., 2008 (link)).
Both collagen and elastin content were normalized to the wet tissue weight for each sample.