Sircol collagen 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.

Total soluble collagen in cell culture supernatants was quantified using the SirCol collagen assay (Biocolor, Belfast, UK) as per the manufacturer’s instructions. Which is an assay based on hydroxyproline. The dermal fibroblast where treated with nothing or IL-13 (100 ng/ml) and after 24 hours the collagen protein was quantitated. Data was normalized to control which was set at 100% collagen.

Total soluble collagen in cell culture supernatants was quantified using the Sircol collagen assay (Biocolor, Belfast, UK). For these experiments, confluent cells in 6-plate wells were incubated for 24 h with 5 ng/mL of TGF-β (Sigma). One mL of Sirius red stain, an anionic dye that reacts specifically with basic collagen side chain groups, was added to 400 μL of supernatant and incubated with gentle rotation for 30 min at room temperature. After centrifugation at 12,000 g for 10 min, the collagen-bound dye was dissolved again after the addition of 1 mL of 0.5 M NaOH and absorbance at 540 nm was measured using a microplate spectrophotometer reader (Synergy HT, BioTek). The absorbance was directly proportional to the amount of newly formed collagen in the cell culture supernatant.

Production of secreted collagen by SELMs was measured with the use of a commercially available Sircol collagen assay (Biocolor, Carrickfergus, UK) according to the manufacturer’s instructions. In brief, 200 μL of ice-cold collagen concentration and isolation reagent were added to 1 mL of cell culture supernatant from each experimental condition and incubated overnight in ice. Samples were then centrifuged, and 1 mL of Sircol dye reagent per sample was added, followed by a 30-min incubation on a mechanical shaker. After centrifugation, the visible collagen pellet was washed with 750 μL of ice-cold acid-salt wash reagent, and the collagen-bound dye was then released by the addition of 250 μL alkali reagent. The ODs of samples and reaction standards were measured in a microplate reader (Diareader EL×800; Dialab, Wr. Neudorf, Austria) at 540 nm against the OD of fresh culture medium as a blank. Collagen concentration was calculated using the linear curve generated by the ODs of the reaction standards.

The total soluble collagen in the Hs68 cell culture supernatant was quantified using the Sircol collagen assay (Biocolor, Belfast, UK). UVB-exposed Hs68 cells were incubated for another 44 h in the dark after Ce6-PEG-Cur-mediated PDT. Following this, 1 mL Sirius red dye, an anionic dye that reacts specifically with the basic side chain groups of collagens under the assay conditions, was added to 400 μL of cell culture medium supernatant and incubated with gentle rotation for 30 min at room temperature. After centrifugation, the pellet was washed with ice-cold acid-salt wash reagent, released in alkali reagent, and the absorbance at 570 nm was measured using an ELISA reader (Sunrise, Tecan, Männedorf, Switzerland). The amount of collagen was calculated based on a standard curve obtained with the standard bovine type Ⅰ collagen supplied with the kit.

The amount of soluble collagen in cell culture supernatants was quantified using the SirCol collagen assay (Biocolor, Belfast, Northern Ireland). The total collagen content of tissue samples was determined by hydroxyproline assays using the chloramines-T method^{28 (link),63 (link)}. In brief, samples were digested with 6 M HCl for 4–6 h. Samples were centrifuged to remove debris and pH of the solution is adjusted to 7. Samples were hydrolyzed by incubation at 60 °C for 30 min. The cloramines-T was added to the hydrolyzate to allow oxidation followed by the addition of Ehrlich’s aldehyde reagent. The absorbance intensity of each sample was analyzed at 550 nm using a microtiter plate reader spectrophotometer.

Total soluble collagen content in the lung lysates was assessed using Sircol collagen assay (Biocolor, UK) according to the manufacturer’s protocol as per34 (link). Collagen assay was performed by mixing lung homogenates with Sircol Dye reagent and measuring absorbance using a plate reader. Collagen content was quantified using a standard curve generated by reference standards and was normalized to the total lung protein content in each sample.