To quantify the sulfated glycosaminoglycan (sGAG) content synthesized by NP cells, the 1,9-dimethylmethylene blue (DMMB) assay was performed as previously described [48 (link)] using a Blyscan sGAG Assay kit (Biocolor Ltd., Carrickfergus, UK) according to the manufacturer's instructions.
Blyscan sgag assay kit
Manufactured by Biocolor
Sourced in United Kingdom
The Blyscan sGAG assay kit is a laboratory tool designed to quantify the presence of sulfated glycosaminoglycans (sGAGs) in various biological samples. It provides a reliable and standardized method for researchers to measure the levels of these important extracellular matrix components.
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Lab products found in correlation
Papain extraction reagent, by Merck Group (3 mentions)
Sircol collagen assay, by Biocolor (2 mentions)
Papain solution, by Merck Group (2 mentions)
Papain, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Spectramax spectrophotometer, by Molecular Devices (1 mentions)
Softmax pro, by Molecular Devices (1 mentions)
Versamax spectrophotometer, by Molecular Devices (1 mentions)
Sircol collagen assay s1000, by Biocolor (1 mentions)
Elastase, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
Sirius red collagen detection kit, by Chondrex (1 mentions)
Dhyal0, by R&D Systems (1 mentions)
Spectramax m2 spectrophotometer, by Molecular Devices (1 mentions)
Mmp 13 elisa kit, by Cusabio (1 mentions)
Dna quantitation kit, by Merck Group (1 mentions)
Synergy ht spectrophotometer, by Agilent Technologies (1 mentions)
Dna quantification kit, by Thermo Fisher Scientific (1 mentions)
19 protocols using blyscan sgag assay kit
1
Quantifying Sulfated Glycosaminoglycan Content
2
Quantification of Sulfated Glycosaminoglycans
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The content of sulfated GAGs was quantified using a Blyscan™ sGAG Assay Kit (B1000; Biocolor, Carrickfergus, UK). The samples were minced into small pieces and digested with the papain extraction reagent (Sigma-Aldrich, St. Louis, MO, USA), L-Cysteina-HCl (Sigma-Aldrich, St. Louis, MO, USA), sodium acetate (Sigma-Aldrich, St. Louis, MO, USA) and EDTA (Sigma-Aldrich, St. Louis, MO, USA) at 65 °C for 18 h. For the DNA quantification, the QuanticoTM PicoGreenTM dsDNA Assay Kit (Thermo Fischer Scientific Nunc, Langenselbold, Germany) was used. The fluorescence was read on a plate reader (Biotek) at an excitation length of 485 nm and an emission of 520 nm.
3
DMMB Assay for Glycosaminoglycan Quantification
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The DMMB (1,9-dimethylmethylene blue) assay was performed using a Blyscan™ sGAG assay kit (Biocolor, B1000, Biocolor, Carrickfergus, UK) according to the manufacturer's instructions. Briefly, harvested tissues and NP cells were washed with PBS, and digested with 1mL papain extraction reagent for 24 h at 65°C. CS contents were determined by reaction with DMMB, and staining was quantified by measuring absorbance at 656 nm. Chondroitin-4-sulfate was used as the standard.
4
Quantification of Skin sGAG Content
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The amount of sulphated glycosaminoglycans (sGAG) was measured by a quantitative dye‐binding method in two separate series. Freeze‐dried skin samples were digested as described in detail previously by Sagstad et al. (2015 (link)), before the concentration of sGAGs was measured using the Blyscan sGAG assay kit (BC‐B1000 or B‐1000; Biocolor, Carrickfergus, UK) in accordance with the manufacturer's instructions. The procedure is based on 1.9‐dimethylmethylene blue that provides a specific binding of the sulphated polysaccharide component of proteoglycans or the protein free sulphated glycosaminoglycan chains. Before analysis, the skin samples were defatted using a chloroform and methanol mixture (400 μL of chloroform and 200 μL of methanol), left in a gentle shaker for 24 h, then 150 μL of chloroform and 150 μL of ultrapure water were added for 2−4 h, followed by drying until a stable weight was attained before being used in the assay. The absorbance values at a wavelength of 650 nm were calculated using the SPECTRAmax® Spectrophotometer and SoftMax Pro (Molecular Devices, San Jose, CA, USA).
5
Quantifying sGAG in Brain Tissues
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The sulfated glycosaminoglycan (sGAG) content
of native and decellularized brains was quantified by using the Blyscan
sGAG Assay Kit (Biocolor, U.K.), following the manufacturer’s
protocol. Briefly, 20 mg of wet tissue samples was weighed and digested
in papain extraction reagent containing 100 μg/mL papain at
65 °C overnight. Blyscan dye reagent was added, and the precipitated
sGAG-dye complexes were dissolved with the dissociation reagent. The
absorbance values of standards and samples at 656 nm were measured
using a microplate reader. Absorbance values were normalized to sample
weight.
of native and decellularized brains was quantified by using the Blyscan
sGAG Assay Kit (Biocolor, U.K.), following the manufacturer’s
protocol. Briefly, 20 mg of wet tissue samples was weighed and digested
in papain extraction reagent containing 100 μg/mL papain at
65 °C overnight. Blyscan dye reagent was added, and the precipitated
sGAG-dye complexes were dissolved with the dissociation reagent. The
absorbance values of standards and samples at 656 nm were measured
using a microplate reader. Absorbance values were normalized to sample
weight.
6
Quantifying Tissue-Derived Sulfated Glycosaminoglycans
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The pericardium was digested with papain solution at 60°C for 6 h. papain
(Sigma, St. Louis, MO) was dissolved at 400 mg/mL in 0.1 M phosphate buffer
(pH 6.0) with 5 mM cysteine hydrochloride and 5 mM EDTA. The lysates were
used for detection of the sGAG amount. The amount of sGAG was measured using
a Blyscan sGAG assay kit (Biocolor, Newtownabbey, UK) according to the
manufacturer's manual. The tissue lysate was mixed with Blyscan dye to bind
the GAG. The GAG-dye complex was then collected by centrifugation.
Subsequently, the supernatant was removed and the tube drained, and the
dissociation reagent was added. Then solution was transferred into a 96-well
plate. Absorbance against the background control was obtained at a
wavelength of 656 nm on a Versamax spectrophotometer (Molecular Devices,
Sunnyvale, USA). The sGAG amount was calculated based on a standard curve
obtained with the standard sGAG supplied with the kit.
(Sigma, St. Louis, MO) was dissolved at 400 mg/mL in 0.1 M phosphate buffer
(pH 6.0) with 5 mM cysteine hydrochloride and 5 mM EDTA. The lysates were
used for detection of the sGAG amount. The amount of sGAG was measured using
a Blyscan sGAG assay kit (Biocolor, Newtownabbey, UK) according to the
manufacturer's manual. The tissue lysate was mixed with Blyscan dye to bind
the GAG. The GAG-dye complex was then collected by centrifugation.
Subsequently, the supernatant was removed and the tube drained, and the
dissociation reagent was added. Then solution was transferred into a 96-well
plate. Absorbance against the background control was obtained at a
wavelength of 656 nm on a Versamax spectrophotometer (Molecular Devices,
Sunnyvale, USA). The sGAG amount was calculated based on a standard curve
obtained with the standard sGAG supplied with the kit.
7
Quantification of Collagen and sGAG Content
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Collagen content was quantified as described previously with a Sircol Collagen Assay (S1000; Biocolor, Carrickfergus, U.K., http://www.biocolor.co.uk ). Briefly, parts of the 4‐ or 12‐weeks samples were cut, minced into 1‐mm3 pieces, and then digested with pepsin (1 mg/ml in 0.5 M acetic acid; MP, Biomedicals, Santa Ana, CA, http://www.mpbio.com/ ), the obtained supernatant was used for testing samples according to kit instructions. Collagen standard curve was calculated, from which total collagen per construct wet weight was obtained.
Sulfated glycosaminoglycans (sGAGs) quantification was processed by using a Blyscan sGAG Assay Kit (B1000; Biocolor) as described elsewhere21 . Briefly, sGAG was extracted from tiny pieces of specimens at 4 or 12 weeks after injection after being digested with papain extraction (Sigma‐Aldrich) reagent at 65°C for 18 hours. Measurement of sGAG concentration in the supernatant was then performed according to the kit instructions, followed by calculation of sGAG per wet weight construct from the sGAG standard curve.
Sulfated glycosaminoglycans (sGAGs) quantification was processed by using a Blyscan sGAG Assay Kit (B1000; Biocolor) as described elsewhere
8
Quantifying sGAG in Lung Tissues
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sGAG content
in native and decellularized lung tissues were assessed by Blyscan
sGAG Assay kit (Biocolor, UK) following manufacturer’s instructions.
Briefly, 15–20 mg of wet tissue samples was digested in a solution
containing 125 μg/mL of papain buffer (400 mg of sodium acetate,
200 mg of EDTA, and 40 mg of cysteine in 50 mL of 0.2 M sodium phosphate
buffer, pH 6.4) at 65 °C overnight. Then, samples were mixed
with the dye reagent, followed by dye retrieval and absorbance measurement
at 656 nm in a microplate reader. sGAG content was normalized to wet
weight of the tissues.
in native and decellularized lung tissues were assessed by Blyscan
sGAG Assay kit (Biocolor, UK) following manufacturer’s instructions.
Briefly, 15–20 mg of wet tissue samples was digested in a solution
containing 125 μg/mL of papain buffer (400 mg of sodium acetate,
200 mg of EDTA, and 40 mg of cysteine in 50 mL of 0.2 M sodium phosphate
buffer, pH 6.4) at 65 °C overnight. Then, samples were mixed
with the dye reagent, followed by dye retrieval and absorbance measurement
at 656 nm in a microplate reader. sGAG content was normalized to wet
weight of the tissues.
9
Quantification of Sulfated Glycosaminoglycans
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Pellet cultures were digested overnight at 60 °C with 300 μg/ml papain (Millipore Sigma, St. Louis, MO, USA) in 20 mM sodium phosphate buffer (pH 6.8) containing 5 mM EDTA and 2 mM dithiothreitol (DTT). Cell lysates were clarified by centrifugation and sGAG was determined using the Blyscan™ sGAG assay kit (Biocolor Ltd, Carrickfergus, UK) according to the manufacturer’s protocol. Briefly, cell lysates were incubated with the 1,9-dimethylmethylene blue (DMMB) dye reagent for 30 min and unbound dye was removed by centrifugation. The bound dye was dissociated from the sGAG–dye complex and quantified by spectrophotometry based on A656. Using chondroitin 4-sulfate as a standard, total sGAG was determined and expressed as a function of the protein content.
10
Quantifying sGAG and COL2 in Tissue Samples
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Samples were first digested with 10 mg/ml of pepsin (Sigma-Aldrich) in 0.05 M acetic acid, followed by digestion with 1 mg/ml of elastase (Sigma-Aldrich). sGAG was quantified using the Blyscan sGAG assay kit (Biocolor, UK). The absorbance of sGAG in the samples was measured at 656 nm, and its concentration was extrapolated from the sGAG standard curve. Type II collagen (COL2) was measured via a captured enzyme-linked immunosorbent assay (ELISA; Chondrex, USA). Absorbance was measured at 490 nm, and the concentration of COL2 in samples was extrapolated from the COL2 standard curve. The total abundance of sGAG and COL2 were normalized to the total DNA content of respective samples, quantified by PicoGreen DNA assay.
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