The Sircol colorimetric assay (Biocolor, Carrickfergus, United Kingdom) was used to evaluate the collagen content. According to the assay’s protocol, we measured 100 mg dry weight of each sample and transferred to low-protein-binding 1.5-mL conical microcentrifuge tubes with 1.0 mL of pepsin (Sigma-Aldrich, St Louis, MO), with a concentration of 0.1 mg/mL of 0.5 M acetic acid to solubilize the collagen by overnight incubation. The collagen content in each sample was evaluated by assessing the absorbance intensity at 555 nm after dye binding according to the manufacturer’s protocol.
Sircol colorimetric assay
Manufactured by Biocolor
Sourced in United Kingdom
The Sircol colorimetric assay is a laboratory equipment designed to quantify soluble collagen. It utilizes a dye-binding method to measure the concentration of collagen in a sample.
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Lab products found in correlation
4 protocols using sircol colorimetric assay
1
Colorimetric Collagen Quantification
2
Quantifying Fibrillar Collagen in Tissues
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Non-crosslinked fibrillar collagen in lung and skin samples was measured by the Sircol colorimetric assay (Biocolor, Belfast, UK). Minced tissues were homogenized in 0.5 M acetic acid with about 1:10 ratio of pepsin (Sigma, USA). Tissues were weighted and incubated overnight at 4 °C with vigorously stirring. Digested samples were centrifuged and the supernatant was used for Sircol dyeing to detect the collagen content. The total protein concentration was determined by using Bradford protein assay kit (Abcam) and the collagen content of each sample was normalized to total protein.
3
Comprehensive Molecular Profiling of Cardiac Stromal Cells
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For Western blotting analyses, cells were lysed in standard sample buffer and run (30 µg) into 4% to 12% gradient SDS polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes followed by incubation with the indicated primary and secondary antibodies (see Major Resource Table ).
For RNA-sequencing analysis, total RNA was extracted from 6 independent cSt-Cs cultured ±TGF-β ±VTP using TRIzol.
After quality checking and quantification, the Poly(A) + RNA was enriched and then processed for RNAseq. Differential gene expression analysis was performed using the R software.
Validation was performed in independent RNA samples of cSt-Cs by RT-qPCR. Indications about the primers sequences and reagents are provided in the Major Resource Table . The effects of TGF-β1±VTP on cSt-Cs contraction were determined using a cell contraction assay kit (Cell Biolabs), as per the Manufacturer's instructions. Collagen plug areas were measured using ImageJ software. Soluble collagen release was quantified using Sircol colorimetric assay (Biocolor) on conditioned medium of cSt-Cs. A full description of the methods employed in molecular analyses is provided in the online supplementary material.
For RNA-sequencing analysis, total RNA was extracted from 6 independent cSt-Cs cultured ±TGF-β ±VTP using TRIzol.
After quality checking and quantification, the Poly(A) + RNA was enriched and then processed for RNAseq. Differential gene expression analysis was performed using the R software.
Validation was performed in independent RNA samples of cSt-Cs by RT-qPCR. Indications about the primers sequences and reagents are provided in the Major Resource Table . The effects of TGF-β1±VTP on cSt-Cs contraction were determined using a cell contraction assay kit (Cell Biolabs), as per the Manufacturer's instructions. Collagen plug areas were measured using ImageJ software. Soluble collagen release was quantified using Sircol colorimetric assay (Biocolor) on conditioned medium of cSt-Cs. A full description of the methods employed in molecular analyses is provided in the online supplementary material.
4
Elastin and Collagen Quantification
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