The collagen content in the resultant LEM and intact liver was measured according to Namiri et al. (2018[32 (link)]) using colorimetric Blyscan assay kits (Biocolor, Carrickfergus, UK). Briefly, the samples were digested with 0.1 M HCl‐pepsin and incubated in a solution that included Sirius red dye. Collagen content was defined by measuring the absorbance at 555 nm (WPA Biowave II, Cambridge, UK).
Blyscan assay kit
Manufactured by Biocolor
Sourced in United Kingdom
The Blyscan assay kit is a laboratory equipment used to quantify the amount of sulfated glycosaminoglycans (sGAG) present in a sample. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Papain, by Merck Group (6 mentions)
Pepsin, by Merck Group (3 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (3 mentions)
Sircol, by Biocolor (2 mentions)
Sircol assay kit, by Biocolor (2 mentions)
Picogreen dsdna reagent, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Quant it picogreen dsdna kit, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
L proline, by Fujifilm (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Ascorbate 2 phosphate, by Merck Group (1 mentions)
Its premix, by Corning (1 mentions)
Elh timp2, by RayBiotech (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Hyaluronidase, by Thermo Fisher Scientific (1 mentions)
Sirt1, by Abcam (1 mentions)
Cleaved caspase 1, by Abcam (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
24 protocols using blyscan assay kit
1
Colorimetric Collagen Content Quantification
2
Quantification of Nerve Extracellular Matrix
3
Quantifying Glycosaminoglycan Production
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Alginate gel beads from days 14 and 28 were washed with PBS and crushed. The samples were digested in papain buffer overnight at 60 °C. The digested samples were subjected to biochemical analyses to determine the glycosaminoglycan content.
Glycosaminoglycan production was determined by using Blyscan assay kit (Biocolor, arrickfergus, Northern Ireland). Glycosaminoglycan content was determined using a standard curve drawn using standard solutions containing chondroitin 4-sulfate. Alginate gel beads without cells were analyzed in the same manner and the values are used as blank.
Glycosaminoglycan production was determined by using Blyscan assay kit (Biocolor, arrickfergus, Northern Ireland). Glycosaminoglycan content was determined using a standard curve drawn using standard solutions containing chondroitin 4-sulfate. Alginate gel beads without cells were analyzed in the same manner and the values are used as blank.
4
Quantification of Collagen, GAG, and DNA in Cell Cultures
5
Biochemical Analysis of Larynx Composition
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Biochemical analysis of native and decellularized canine larynxes were performed to determine the presence of glycosaminoglycan (GAG) and double-stranded DNA (dsDNA).
Samples were divided in 3 groups: cartilage (n=3), muscle (n=3) and mucosa/submucosa (n=3). Glycosaminoglycans (GAGs) analysis was performed using the Blyscan assay kit (Biocolor, UK). Dry samples were homogenized in 1 ml of papain buffer containing 7 μl / ml of papain, digested at 65°C and incubated with the dye reagent. After washing, the samples were read in a 96-well plate, at 656nm and concentrations were calculated in reference to the relative standard curve. For dsDNA quantification, tissue samples were digested overnight at 55 °C in 310 μL of a solution containing 10 mM Tris (pH = 8.0), 5 mM EDTA, 0.1 M NaCl, 1% SDS, and 650 μg/mL Proteinase-K (Life Technologies, Carlsbad, CA).
dsDNA was then isolated using a phenol/chloroform extraction and alcohol precipitation, and quantified using the Quant-iT PicoGreen dsDNA kit (Life Technologies) following the manufacturer protocols. Fluorescence was read at 480nm/520nm (excitation/emission) and concentrations were calculated in relation to the standard curve. All biochemical data are presented as the total mass of the component extracted normalized to tissue sample wet weight and normalized to dry weight (lyophilized tissue).
Samples were divided in 3 groups: cartilage (n=3), muscle (n=3) and mucosa/submucosa (n=3). Glycosaminoglycans (GAGs) analysis was performed using the Blyscan assay kit (Biocolor, UK). Dry samples were homogenized in 1 ml of papain buffer containing 7 μl / ml of papain, digested at 65°C and incubated with the dye reagent. After washing, the samples were read in a 96-well plate, at 656nm and concentrations were calculated in reference to the relative standard curve. For dsDNA quantification, tissue samples were digested overnight at 55 °C in 310 μL of a solution containing 10 mM Tris (pH = 8.0), 5 mM EDTA, 0.1 M NaCl, 1% SDS, and 650 μg/mL Proteinase-K (Life Technologies, Carlsbad, CA).
dsDNA was then isolated using a phenol/chloroform extraction and alcohol precipitation, and quantified using the Quant-iT PicoGreen dsDNA kit (Life Technologies) following the manufacturer protocols. Fluorescence was read at 480nm/520nm (excitation/emission) and concentrations were calculated in relation to the standard curve. All biochemical data are presented as the total mass of the component extracted normalized to tissue sample wet weight and normalized to dry weight (lyophilized tissue).
6
Quantification of Collagen, GAGs, and DNA
7
Quantifying sGAG in 3D Cell Spheroids
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Spheroid samples were digested overnight at 65 °C in 100 µL of papain digestion buffer (1 mg/mL) (containing 2 mM Acetyl Cysteine and 2 mM EDTA in 50 mM sodium phosphate) (Sigma). The supernatant was collected by centrifugation for sGAG quantification, using the Blyscan Assay kit (Biocolor, Carrickfergus, Co Antrim, UK), according to the manufacturer’s protocol. sGAG levels in the samples were calculated from a linear standard curve of chondroitin-4-sulfate, range 0–5 µg of sGAGs.
8
Chondrogenic Differentiation of Cell Pellets
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To obtain cell aggregated pellets, 2 × 105 cells were centrifuged in a 96-deep well polypropylene plates and cultured with 10% FBS-aMEM. The next day, the medium was changed to a chondrogenic medium that consisted of high-glucose DMEM containing 110 μg/mL sodium pyruvate (Gibco) supplemented with 0.2 mM ascorbate-2-phosphate (Sigma-Aldrich), 40 μg/mL L-proline (Wako), 100 nM dexamethasone (Sigma-Aldrich), 1% ITS + Premix (Corning: 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 μg/mL selenious acid, 1.25 μg/mL bovine serum albumin, and 5.35 μg/mL linoleic acid), 10 ng/mL transforming growth factor-β3 (TGFβ3) (Peprotech, Rocky Hill, New Jersey, USA), and 50 ng/mL bone morphogenic protein 2 (BMP2) (Medtronic, Dublin, Ireland). The pellets were maintained with 0.5 mL of chondrogenic culture medium in a humid atmosphere of 5% CO2 at 37°C. The medium was replaced twice per week.
Total sulfated glycosaminoglycan (sGAG) was measured using a Blyscan Assay Kit (Biocolor, Westbury, NY, USA) based on 1,9-dimethylmethylene blue binding against a standard curve of chondroitin-6-sulfate according to the manufacturer's protocol.
Total sulfated glycosaminoglycan (sGAG) was measured using a Blyscan Assay Kit (Biocolor, Westbury, NY, USA) based on 1,9-dimethylmethylene blue binding against a standard curve of chondroitin-6-sulfate according to the manufacturer's protocol.
9
Chondrocyte Viability and Glycosaminoglycan Analysis
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After 72 hours of co-culture, the microspheres solution was discarded, and the chondrocytes were removed to a fresh media. After standardizing cell samples to one million in different groups using a cell counter, 0.5 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-dippphenyltetrazolium bromide (MTT) was added to chondrocytes incubated at 37°C with 5% CO2 for 4 h. The resulting formation was dissolved in dimethylsulphoxide and absorbance was measured at 570 nm with a microplate reader.
The GAG content in cell supernatants was assessed using Blyscan assay kit (Biocolor, UK), and the chondrocytes were normalized to one million cells in each group. Briefly, cell supernatants were digested enzymatically using proteinase K. Following digestion, a desired amount of supernatant was reacted with Blyscan dye for 30 min. GAG-dye precipitate was obtained by centrifugation and the resulting formation was dissolved in 1 ml dissociation reagent and incubated for 10 minutes. Finally, samples were transferred to a 96-well-plate and absorbance at 656 nm was measured on the spectrophotometer. A standard curve was derived from mixed-isomer shark chondroitin sulfate, and the GAG content was calculated.
The GAG content in cell supernatants was assessed using Blyscan assay kit (Biocolor, UK), and the chondrocytes were normalized to one million cells in each group. Briefly, cell supernatants were digested enzymatically using proteinase K. Following digestion, a desired amount of supernatant was reacted with Blyscan dye for 30 min. GAG-dye precipitate was obtained by centrifugation and the resulting formation was dissolved in 1 ml dissociation reagent and incubated for 10 minutes. Finally, samples were transferred to a 96-well-plate and absorbance at 656 nm was measured on the spectrophotometer. A standard curve was derived from mixed-isomer shark chondroitin sulfate, and the GAG content was calculated.
10
Quantification of Inflammatory Markers in Intervertebral Disc Culture
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The concentration of PGE 2 (K051-H5, Arbor Assays, Ann Arbor, MI, USA), bIL-6 (MBS9141101, MyBioSource San Diego, CA, USA), hIL-6 (430507, BioLegend), hIL-1β (DLB50, R&D Systems), hIL-1ra (BRA00B, R&D Systems), hCFH (ab137975, Abcam), hTIMP-1 (ELH-TIMP1, RayBiotech, Peachtree Corners, GA, USA) and hTIMP-2 (ELH-TIMP2, RayBiotech) was determined by ELISA in the supernatants at days 11 and 16 of organ culture.
DNA and protein quantification in the AF tissue AF tissues were digested overnight at 56 °C using 0.5 mg/mL proteinase K (P6556, Sigma-Aldrich) solution for DNA and sGAG quantification. DNA content was determined using the Quant-iT PicoGreen dsDNA assay kit (P7589, Invitrogen). sGAG content was determined using the Blyscan assay kit (B1000, Biocolor, Carrickfergus, UK). AF tissues were digested for soluble collagen and elastin quantification according to the Sircol (S1000, Biocolor) and Fastin (F2000, Biocolor) assay kits, respectively.
DNA and protein quantification in the AF tissue AF tissues were digested overnight at 56 °C using 0.5 mg/mL proteinase K (P6556, Sigma-Aldrich) solution for DNA and sGAG quantification. DNA content was determined using the Quant-iT PicoGreen dsDNA assay kit (P7589, Invitrogen). sGAG content was determined using the Blyscan assay kit (B1000, Biocolor, Carrickfergus, UK). AF tissues were digested for soluble collagen and elastin quantification according to the Sircol (S1000, Biocolor) and Fastin (F2000, Biocolor) assay kits, respectively.
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