Bovine chondroitin sulfate

Manufactured by Merck Group

Bovine chondroitin sulfate is a naturally occurring polysaccharide derived from bovine cartilage. It is a component of the extracellular matrix and plays a role in the structure and function of cartilage and other connective tissues.

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Chondroitin sulfate (134 citations) Chondroitin sulfate A from bovine trachea (4 citations) Chondroitin sulfate A sodium salt from bovine trachea (4 citations) Chondroitin sulfate from bovine trachea (4 citations) Chondroitin sulfate sodium salt from bovine cartilage (3 citations) Chondroitin-4-sulfate from bovine trachea (3 citations) Bovine chondroitin-4-sulfate (2 citations)

9 protocols using bovine chondroitin sulfate

Quantifying cell density and GAG

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Representative pellets (n = 4) were digested at 60 °C for 4 h with 125 μg/mL papain in PBE buffer (100 mM phosphate, 10 mM ethylenediaminetetraacetic acid, pH 6.5) containing 10 mM cysteine. To quantify cell density, the amount of DNA in the papain digestion was measured using the QuantiT™ PicoGreen^® dsDNA assay kit (Life Technologies) with a CytoFluor^® Series 4000 (Applied Biosystems, Foster City, CA). GAG was measured using dimethylmethylene blue dye and a Spectronic™ BioMate™ 3 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) with bovine chondroitin sulfate (Sigma–Aldrich) as a standard.

Li J., He F, & Pei M. (2015). Chondrogenic priming of human fetal synovium-derived stem cells in an adult stem cell matrix microenvironment. Genes & Diseases, 2(4), 337-346.

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Extracellular Matrix Quantification

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Representative cell/scaffold constructs (n=3) in each group were crushed as described above. Then the mixture was digested by papain solution (125 μg/mL papain, 100 mM phosphate, 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM cysteine, pH 6.3) at 60°C overnight. The GAG content was determined via dimethylmethylene blue (DMMB, Sigma-Aldrich) assay to read the absorbance at 525 nm with bovine chondroitin sulfate (Sigma) as a standard. The GAG content was normalized by total DNA that was measured via Hoechst 33258 assay with calf thymus DNA (Sigma-Aldrich) as a standard.

Liu Q., Wang J., Chen Y., Zhang Z., Saunders L., Schipani E., Chen Q, & Ma P.X. (2018). Suppressing Mesenchymal Stem Cell Hypertrophy and Endochondral Ossification in 3D Cartilage Regeneration with Nanofibrous Poly(L-Lactic Acid) Scaffold and Matrilin-3. Acta biomaterialia, 76, 29-38.

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Quantifying Cell Density and GAG Content

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The pellets (n=4) were digested for 4 h at 60°C with 125 μg/mL papain in PBE buffer (100 mM phosphate, 10 mM ethylenediaminetetraacetic acid, pH 6.5) containing 10 mM cysteine. To quantify cell density, the amount of DNA in the papain digestion was measured using the QuantiT™ PicoGreen® dsDNA assay kit (Invitrogen, Carlsbad, CA) with a CytoFluor® Series 4000 (Applied Biosystems, Foster City, CA). GAG was measured using dimethylmethylene blue dye and a Spectronic™ BioMate™ 3 Spectrophotometer (ThermoFisher Scientific) with bovine chondroitin sulfate (Sigma-Aldrich) as a standard.

Li J., Narayanan K., Zhang Y., Hill R.C., He F., Hansen K.C, & Pei M. (2019). Role of lineage-specific matrix in stem cell chondrogenesis. Biomaterials, 231, 119681.

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Cartilage Extracellular Matrix Analysis

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MSC-seeded scaffolds and meniscus repair model inner cores were
digested in 0.5 mL or 1.5 mL, respectively, of 125 μg/mL papain
overnight at 65°C^{54 (link),
67 , 68 (link)}. DNA content was measured using
the picogreen assay (Invitrogen, Carlsbad, CA). Sulfated
glycosaminoglycan (sGAG) content was measured using the
dimethylmethylene blue assay^{69 (link)} and bovine chondroitin sulfate as a standard
(Sigma-Aldrich). Collagen content was assessed with the hydroxyproline
(OHP) assay^{70 (link)}, using
trans-4-hydroxy-L-proline (Sigma-Aldrich) as a standard, as previously
described^{54 (link), 67 , 68 (link)}.

Hidalgo Perea S., Lyons L.P., Nishimuta J.F., Weinberg J.B, & McNulty A.L. (2019). Evaluation of Culture Conditions for In vitro Meniscus Repair Model Systems using Bone Marrow-Derived Mesenchymal Stem Cells. Connective tissue research, 61(3-4), 322-337.

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Quantification of GAG Content in Samples

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GAG content in culture supernatants was assessed using the Barbosa method [40 (link)]. Briefly, 250 μL of collected supernatant was incubated with 1 mL of DMMB solution (16 mg/l dimethylmethylene blue, 6 mM sodium formate, 200 mM GuHCL, all from Sigma Aldrich, pH 3.0) on a shaker at room temperature for 30 min. After centrifugation, precipitated DMMB-GAG complexes were dissolved in decomplexion solution (4 M GuHCL, 50 mM Na-Acetate, 10% Propan-1-ol, all from Sigma Aldrich, pH 6.8) at 60 °C for 15 min. Absorption was measured at 656 nm and corresponding GAG concentrations were calculated using a standard curve prepared with purified bovine chondroitin sulfate (Sigma Aldrich).
For the measurement of GAG content in cartilage tissue, samples were preliminary digested overnight at 56 °C in 1 mL of proteinase K solution (Sigma Aldrich, P2308), and 100 µL of the resulting digested solution was used for the DMMB-GAG precipitation.

Pigeot S., Bourgine P.E., Claude J., Scotti C., Papadimitropoulos A., Todorov A., Epple C., Peretti G.M, & Martin I. (2020). Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage. International Journal of Molecular Sciences, 21(19), 7233.

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Glycosaminoglycan Quantification Protocol

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Culture media were collected at 48 h post-CTS. Bimethylmethylene blue dye (DMMB) (Sigma) was used to spectrophotometrically quantify the sulfated glycosaminoglycan. Bovine chondroitin sulfate (Sigma) served as standard controls [25 (link)].

Chen C., Wei X., Lv Z., Sun X., Wang S., Zhang Y., Jiao Q., Wang X., Li Y, & Wei L. (2016). Cyclic Equibiaxial Tensile Strain Alters Gene Expression of Chondrocytes via Histone Deacetylase 4 Shuttling. PLoS ONE, 11(5), e0154951.

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Quantification of GAGs and DNA in Hydrogels

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For the biochemical
assessment of glycosaminoglycans (GAGs) and DNA content, hydrogels
were digested overnight at 65 °C inside papain buffer solution
(pH = 6.5), containing 100 mM Na₂HPO₄, 10 mM l-cysteine, 10 mM EDTA, and 6 μL ml^–1 papain enzyme (Sigma-Aldrich). The GAG content was determined using
the 1,9-dimethylmethylene blue assay, at pH 1.5. Sample absorbance
was measured at 530 and 590 nm and compared to standard curve of bovine
chondroitin sulfate (Sigma-Aldrich). DNA content was quantified using
Hoechst 33258 DNA intercalating dye method (ThermoFisher Scientific),
and purified Calf Thymus DNA was used as the standard.

Stampoultzis T., Guo Y., Nasrollahzadeh N., Karami P, & Pioletti D.P. (2023). Mimicking Loading-Induced Cartilage Self-Heating in Vitro Promotes Matrix Formation in Chondrocyte-Laden Constructs with Different Mechanical Properties. ACS Biomaterials Science & Engineering, 9(2), 651-661.

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Quantifying DNA and Glycosaminoglycans

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Harvested micromasses and pellets were digested at 56°C overnight with 0.1 M proteinase K in PBE buffer (1 mM EDTA, 10 mM Tris, pH 6.5). DNA amount was measured using the nanodrop (nanodrop 2000). GAG amount was measured at 525 nm with a spectrophotometer (BioTek Synergy) using DMMB dye added to 30 µL of pellet digestion or undiluted supernatant and bovine chondroitin sulfate (Sigma) as a standard.

Guns L.A., Monteagudo S., Kvasnytsia M., Kerckhofs G., Vandooren J., Opdenakker G., Lories R.J, & Cailotto F. (2017). Suramin increases cartilage proteoglycan accumulation in vitro and protects against joint damage triggered by papain injection in mouse knees in vivo. RMD Open, 3(2), e000604.

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Quantifying Cartilage Composition Metrics

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Pellets were rinsed with DPBS, digested overnight at 65°C in 200
μl of papain solution consisting of 125 μg/ml papain, 100 mM
sodium phosphate, 5 mM EDTA, and 5 mM L-cysteine hydrochloride at 6.5 pH, then
stored at −20°C. Double-stranded DNA content was quantified using
the PicoGreen™ assay (Invitrogen, Carlsbad CA). Total sulfated
glycosaminoglycan (s-GAG) content was measured using the dimethymethlyene blue
(DMMB) assay using bovine chondroitin sulfate (Sigma Aldrich) as a standard.
Collagen content was measured using the hydroxyproline assay as previously
described.[61 (link)]

Adkar S.S., Wu C.L., Willard V.P., Dicks A., Ettyreddy A., Steward N., Bhutani N., Gersbach C.A, & Guilak F. (2018). Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing. Stem cells (Dayton, Ohio), 37(1), 65-76.

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