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Chondroitin sulfate c

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Chondroitin sulfate C is a sulfated glycosaminoglycan found in the extracellular matrix of various connective tissues. It is a laboratory-grade product primarily used for research purposes.

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Lab products found in correlation

Spectrofluorometer, by Bio-Rad (3 mentions) Quant it picogreen dsdna kit, by Thermo Fisher Scientific (3 mentions) Papain solution, by Merck Group (2 mentions) Cysteine, by Merck Group (2 mentions) Chondroitin sulfate a, by Merck Group (2 mentions) P3125, by Merck Group (1 mentions) Versamax plate reader, by Molecular Devices (1 mentions) Heparin, by Iduron (1 mentions) Acetone, by Merck Group (1 mentions) Thermomixer, by Eppendorf (1 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Quant it picogreen dsdna quantification assay, by Thermo Fisher Scientific (1 mentions) Hyaluronic acid, by Merck Group (1 mentions) Heparin, by Merck Group (1 mentions) Neuraminidase, by Merck Group (1 mentions) Chondroitinase abc, by Merck Group (1 mentions) Sodium chlorate, by Merck Group (1 mentions) Chondroitin sulfate b, by Merck Group (1 mentions) Heparinase 3, by Merck Group (1 mentions) Indomethacin, by Merck Group (1 mentions)

Spelling variants of this product

Chondroitin sulfate (134 citations) Chondroitin sulfate A sodium salt (CS) (2 citations) Mouse anti-chondroitin sulfate antibody (CS-56, (2 citations)

24 protocols using chondroitin sulfate c

1

Quantification of Proteoglycans and DNA in Cell-Laden Hydrogels

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The remaining halves of the harvested cell-laden hydrogels were used for biochemical analysis. The samples were weighted (wet weight), freeze dried overnight and weighed again (dry weight). To determine the GAG and DNA contents, the samples were digested overnight at 56°C in 200 µL papain digestion buffer (0.2 M NaH2PO4 + 0.01 M EDTA*2 H2O in milliQ, pH = 6.0) supplemented with 250 µL/mL papain solution (16-40 units/mg protein, P3125, Sigma Aldrich) and 0.01 M cysteine (C9768, Sigma Aldrich). The amount of sulfated GAGs, as a measure of proteoglycans, was determined with a dimethylmethylene blue (DMMB, pH = 3.0) assay31 (link) using known concentrations of chondroitin sulfate C (Sigma Aldrich) as a reference. In short, samples were diluted in PBS-EDTA and mixed with the DMMB solution. Excitation was measured directly after mixing at 525 nm and 595 nm with a versa max plate reader (Molecular devices, Wokingham, UK). The measurement at 525 nm was divided by the measurement at 595 nm and the GAG concentration of the samples was calculated from a quadratic fit of the standard curve and were corrected for the dilution. Quantification of DNA was performed with a Quant-iT PicoGreen dsDNA kit (Molecular Probes, Invitrogen) using a spectrofluorometer (Biorad, Veenendaal, the Netherlands).
Mouser V.H., Melchels F.P., Visser J., Dhert W.J., Gawlitta D, & Malda J. (2016). Yield stress determines bioprintability of hydrogels based on gelatin-methacryloyl and gellan gum for cartilage bioprinting. Biofabrication, 8(3), 035003.
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2

Chemically Modified Heparin Derivatives: Synthesis and Characterization

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Heparin (17 kDa average molecular mass, Celsus Lab, Cincinnati, OH) was used in all assays, and as the starting material for the production of modified derivatives and oligosaccharides. Different chemically modified Heparin derivatives D1–9 (table 1; [35 (link)]), and cationic forms were produced, as described [36 (link)], whereas oligosaccharides with degrees of polymerization (dp) dp2–dp12 were obtained from Iduron (Manchester, UK). Porcine mucosal HS, hyaluronic acid (HA, not sulfated) and chondroitin sulfate C (CS-C, average sulfate per disaccharide unit, 1) were from Sigma (Gillingham, Dorset, UK); Dermatan sulfate (DS, average sulfate per disaccharide unit, 1) were from Iduron.

Nomenclature and structures of chemically modified Heparin structures. I stands for iduronate, and A stands for the amino sugar glucosamine. aNumbers refer to the ring position of carbon atoms. The average number of sulfate groups per disaccharide is also indicated.

analoguepredominant repeatIdoUA-2GlcN-6GlcN-2IdoUA-3GlcN-3asulfate groups per disaccharide
D1 (Heparin)I2SA6SNsSO3−SO3−SO3−OHOH2.4
D2I2SA6SNAcSO3−SO3−COCH3OHOH1.8
D3I2OHA6SNsOHSO3−SO3−OHOH1.9
D4I2SA6SNsSO3−OHSO3−OHOH1.8
D5I2OHA6SNAcOHSO3−COCH3OHOH1.2
D6I2SA6OHNAcSO3−OHCOCH3OHOH0.8
D7I2OHA6OHNsOHOHSO3−OHOH0.8
D8I2OHA6OHNAcOHOHCOCH3OHOH0
D9I2S,3SA6S3SNsSO3−SO3−SO3−SO3−SO3−4.4
Li Y., Sun C., Yates E.A., Jiang C., Wilkinson M.C, & Fernig D.G. (2016). Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification. Open Biology, 6(3), 150275.
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3

Quantifying Glycosaminoglycans via DMB Assay

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1,9-dimethylmethylene blue (DMB; Sigma-Aldrich) solution (46 μM DMB, 40 mM glycine, 40 mM NaCl [pH 3.0]) was added to proteinase K-digested samples (200 μl) and the 530:590 nm absorbance ratio was measured to determine the glycosaminoglycan amount, using chondroitin sulfate C (Sigma-Aldrich) as a standard.
Narcisi R., Cleary M.A., Brama P.A., Hoogduijn M.J., Tüysüz N., ten Berge D, & van Osch G.J. (2015). Long-Term Expansion, Enhanced Chondrogenic Potential, and Suppression of Endochondral Ossification of Adult Human MSCs via WNT Signaling Modulation. Stem Cell Reports, 4(3), 459-472.
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4

Quantification of Extracellular Matrix Glycosaminoglycans

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GAG content was determined using dimethylmethylene blue (DMMB, Sigma-Aldrich USA) test [29 ]. A total of 5×103 cells were cultivated on selected scaffolds with the size of 0.5×0.5 cm. After 14 and 21 days, while half of the medium was replaced each three days, 500 μL of medium was taken out for further evaluation. Then 1.5 mL acetone (Merck, Germany) was added to the collected medium and kept at -20°C for 24 h. Samples were centrifuged at 260 ×g at 4°C for 30 min. The precipitated material was suspended in 100 μL PBS containing papain (20 μg/mL), activated with 5 mM cysteine, and followed by incubation at 60°C for 16 h and boiling for 15 min. A working standard solution of chondroitin sulfate C (shark cartilage extract, Sigma-Aldrich, USA) was prepared. The DMMB assays were performed in 96-well plates using an ELISA plate reader and the optical density was immediately measured at 545 nm.
Nematollahi Z., Tafazzoli-Shadpour M., Zamanian A., Seyedsalehi A., Mohammad-Behgam S., Ghorbani F, & Mirahmadi F. (2017). Fabrication of Chitosan Silk-based Tracheal Scaffold Using Freeze-Casting Method. Iranian Biomedical Journal, 21(4), 228-239.
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5

Quantification of GAG and DNA in Constructs

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To quantify retained glycosaminoglycans (GAG) and DNA, cultured constructs were frozen and lyophilized. After careful removal of the cell-free surface hydrogel layer, the cell-laden proportions of the constructs were digested overnight in 0.5 mg/mL proteinase K (Invitrogen) at 56 °C on an Eppendorf® Thermomixer® (Eppendorf, Hamburg, Germany). GAG concentration in the digest was measured using the dimethyl-methylene blue (DMMB) assay (pH 1.5). Absorbances at 525 and 595 nm were measured, and concentrations calculated using the ratio of absorbances, compared to a quadratic standard curve prepared from chondroitin sulfate C (Sigma-Aldrich). DNA concentrations in the digests were measured using the Quant-iT™ PicoGreen® dsDNA quantification assay (Invitrogen).
Meinert C., Schrobback K., Levett P.A., Lutton C., Sah R.L, & Klein T.J. (2016). Tailoring hydrogel surface properties to modulate cellular response to shear loading. Acta biomaterialia, 52, 105-117.
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6

Cartilage Tissue Analysis Protocol

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At days 0 or 1, 29 and 57, the cartilage of the OC plugs was cut in half and the tissue inside the defect area was separated from the surrounding cartilage. Tissue samples were weighed (wet weight), freeze dried, and weighed again (dry weight) to determine the water content. Next, samples were digested overnight at 56°C in 200 μl papain digestion buffer (0.2 M NaH2PO4 + 0.01 M EDTA*2 H2O in milliQ, pH = 6.0) supplemented with 250 μl/ml papain solution (16-40 units/mg protein, Sigma Aldrich) and 0.01 M cysteine (Sigma Aldrich). In addition to tissue samples, medium samples were taken from the cartilage compartment upon each medium change. The amount of sulfated glycosaminoglycans (GAGs), both in the digested tissue and the medium, as a measure of proteoglycans, was determined with a dimethylmethylene blue (DMMB, pH = 3.0) assay (Farndale et al., 1982 (link)), using known concentrations of chondroitin sulfate C (Sigma Aldrich) as a reference. Quantification of DNA was performed with a Quant-iT PicoGreen dsDNA kit (Molecular Probes, Invitrogen, Carlsbad, USA) using a spectrofluorometer (Biorad, Veenendaal, The Netherlands) according to the instructions of the manufacturer.
Mouser V.H., Dautzenberg N.M., Levato R., van Rijen M.H., Dhert W.J., Malda J, & Gawlitta D. (2017). Ex Vivo Model Unravelling Cell Distribution Effect in Hydrogels for Cartilage Repair. ALTEX, 35(1), 65-76.
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7

Glycosaminoglycan Characterization Protocol

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Heparin (from porcine intestinal mucosa, product ID H3149), hyaluronic acid (HA; from Streptococcus equi, product ID 94137), chondroitin sulfate A (CS-A; from bovine trachea, product ID C9819), chondroitin sulfate B (CS-B; from porcine intestinal mucosa, product ID C3788), chondroitin sulfate C (CS-C; from shark cartilage, product ID C4384), sodium chlorate (product ID 244147), Heparinase III (hepIII; from Flavobacterium Heparinum, product ID H8891), neuraminidase (neu; from Vibrio cholerae, product ID N7885), and chondroitinase ABC (ChABC; from Proteus vulgaris, product ID C3667) were purchased from Sigma (St. Louis, USA). Keratan sulfate (from bovine cornea) was a gift from Prof. Robert J Linhardt. Heparin oligosaccharide 14-mer (H014) and CSC-14 mer (CS-C 14-mer; CS014) were purchased from Iduron (Macclesfield, UK). Heparin hexamer (GAG 012) and dimer (GAG 063), and GD1a-glycan were purchased from Elicityl (Crolles, France).
Chandra N., Liu Y., Liu J.X., Frängsmyr L., Wu N., Silva L.M., Lindström M., Chai W., Pedrosa Domellöf F., Feizi T, & Arnberg N. (2019). Sulfated Glycosaminoglycans as Viral Decoy Receptors for Human Adenovirus Type 37. Viruses, 11(3), 247.
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8

Quantifying Cartilage Degradation via S-GAGs

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A massive release of sulfated glycosaminoglycans (S-GAGs) represents cartilage matrix degradation in a pathological condition. Measuring the levels of S-GAGs using dimethylmethylene blue (DMMB) assay in culture media was performed as previously described [46 (link)]. Chondroitin sulfate C (Sigma-Aldrich, St. Louis, MO, USA) was used as the standard for the calculation of S-GAGs concentrations.
Kongdang P., Chokchaitaweesuk C., Tangyuenyong S, & Ongchai S. (2019). Proinflammatory Effects of IL-1β Combined with IL-17A Promoted Cartilage Degradation and Suppressed Genes Associated with Cartilage Matrix Synthesis In Vitro. Molecules, 24(20), 3682.
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9

Quantifying Sulfated GAG Deposition in ADSC Cultures

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The DMMB assay was used to quantify sGAG deposition by ADSCs. DMMB reacts with the sulfate group of the GAG chain and will not react with nonsulfated GAGs such as HA [39 (link)]. At each indicated time point, the DNA content and sGAG deposition of each sample were quantified spectrofluorometrically using Hoechst 33,258 dye and DMMB, respectively [34 (link),36 (link),40 (link),41 (link)]. A standard curve for the DMMB assay was generated using an aqueous solution of chondroitin sulfate C (Sigma-Aldrich, St. Louis, MO, USA) with concentrations ranging from 0 to 25 μg/μL.
Wu S.C., Chang C.H., Chang L.H., Wu C.W., Chen J.W., Chen C.H., Lin Y.S., Chang J.K, & Ho M.L. (2021). Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment. Biomedicines, 9(5), 559.
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10

Cartilage Tissue Analysis Protocol

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At days 0 or 1, 29 and 57, the cartilage of the OC plugs was cut in half and the tissue inside the defect area was separated from the surrounding cartilage. Tissue samples were weighed (wet weight), freeze dried, and weighed again (dry weight) to determine the water content. Next, samples were digested overnight at 56°C in 200 μl papain digestion buffer (0.2 M NaH2PO4 + 0.01 M EDTA*2 H2O in milliQ, pH = 6.0) supplemented with 250 μl/ml papain solution (16-40 units/mg protein, Sigma Aldrich) and 0.01 M cysteine (Sigma Aldrich). In addition to tissue samples, medium samples were taken from the cartilage compartment upon each medium change. The amount of sulfated glycosaminoglycans (GAGs), both in the digested tissue and the medium, as a measure of proteoglycans, was determined with a dimethylmethylene blue (DMMB, pH = 3.0) assay (Farndale et al., 1982 (link)), using known concentrations of chondroitin sulfate C (Sigma Aldrich) as a reference. Quantification of DNA was performed with a Quant-iT PicoGreen dsDNA kit (Molecular Probes, Invitrogen, Carlsbad, USA) using a spectrofluorometer (Biorad, Veenendaal, The Netherlands) according to the instructions of the manufacturer.
Mouser V.H., Dautzenberg N.M., Levato R., van Rijen M.H., Dhert W.J., Malda J, & Gawlitta D. (2017). Ex Vivo Model Unravelling Cell Distribution Effect in Hydrogels for Cartilage Repair. ALTEX, 35(1), 65-76.
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