Pellets were digested at day 21 of chondrogenic differentiation using 1 mg/mL Proteinase K, 1 mM iodoacetamide, 10 μg/mL Pepstatin A in 50 mM Tris, and 1 mM EDTA buffer (pH 7.6; all Sigma-Aldrich) for 16 h at 56°C, followed by Proteinase K inactivation at 100°C for 10 min. Afterward, to determine the amount of DNA, cell lysates were treated with 0.415 IU heparin and 1.25 µg RNase for 30 min at 37°C, followed by the addition of 30 μL CYQUANT GR solution (Invitrogen). The samples were analyzed using a SpectraMax Gemini plate reader with an excitation of 480 nm and an emission of 520 nm. As a standard, DNA sodium salt from calf thymus (Sigma-Aldrich) was used. To determine the amount of GAG, cell lysates were incubated with 1, 9-dimethylmethylene blue (DMB), as previously described by Farndale et al. (1986 (link)), and analyzed with an excitation of 590 nm and 530 nm. The 530:590 nm ratio was used to determine the GAG concentration. As a standard, chondroitin sulfate sodium salt from shark cartilage (Sigma-Aldrich) was used.
Dna sodium salt from calf thymus
Manufactured by Merck Group
Sourced in United States
DNA sodium salt from calf thymus is a purified form of deoxyribonucleic acid (DNA) extracted from the thymus gland of calves. It serves as a source of genetic material for various research and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Pepstatin a, by Merck Group (2 mentions)
Edta buffer, by Merck Group (1 mentions)
Chondroitin sulfate sodium salt from shark cartilage, by Merck Group (1 mentions)
Cyquant cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Infinite 200 reader, by Tecan (1 mentions)
C4384, by Merck Group (1 mentions)
D3664, by Merck Group (1 mentions)
Tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
1 9 dimethylmethylene blue dmb, by Merck Group (1 mentions)
Chondroitin sulphate sodium salt from shark cartilage, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
5 protocols using dna sodium salt from calf thymus
1
Quantifying DNA and GAG in Chondrogenesis
2
Quantifying Scaffold DNA and GAG Content
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DNA and GAG content of the scaffolds (400 µm,r 600 µm or 1 mm in thickness, 6 scaffolds per group) before and after the decell-deGAG treatment were assessed using the CyQUANT® Cell Proliferation Kit (C7026, LifeTechnologies, United States) and the dimethyl methylene blue (DMMB) assay [50] (link) respectively. Twelve untreated human articular cartilage biopsies served as control.
Samples were digested with papain according to a protocol adapted from Kim et al. [51] (link) For DNA quantification, digested samples were incubated with RNAse (R5125, Sigma Aldrich, United States) for 30 min before adding CyQUANT GR dye. Fluorescence was measured at 480ex/520em on a TECAN Infinite 200 reader (Tecan Group AG, Switzerland) in 96-well plates (Greiner, Austria). Measurements were performed in duplicate and DNA content was calculated using linear regression to a standard curve (DNA sodium salt from calf thymus, D3664, Sigma Aldrich, United States).
For the DMMB assay, digested samples were diluted with PBS-EDTA and dimethyl methylene blue was added, resulting in metachromatic changes in absorbance that were detected at 530 and 590 nm. The GAG content was calculated via linear regression to a chondroitin sulphate standard (C4384, Sigma Aldrich, United States).
Samples were digested with papain according to a protocol adapted from Kim et al. [51] (link) For DNA quantification, digested samples were incubated with RNAse (R5125, Sigma Aldrich, United States) for 30 min before adding CyQUANT GR dye. Fluorescence was measured at 480ex/520em on a TECAN Infinite 200 reader (Tecan Group AG, Switzerland) in 96-well plates (Greiner, Austria). Measurements were performed in duplicate and DNA content was calculated using linear regression to a standard curve (DNA sodium salt from calf thymus, D3664, Sigma Aldrich, United States).
For the DMMB assay, digested samples were diluted with PBS-EDTA and dimethyl methylene blue was added, resulting in metachromatic changes in absorbance that were detected at 530 and 590 nm. The GAG content was calculated via linear regression to a chondroitin sulphate standard (C4384, Sigma Aldrich, United States).
3
Enzyme-Linked Immunosorbent Assay (ELISA) Protocol
4
Quantification of DNA and GAG in Cell Lysates
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The pellets were digested using 1 mg/mL Proteinase K, 1 mM iodoacetamide, 10 µg/mL Pepstatin A in 50 mM Tris, 1 mM EDTA buffer (250 μL) (pH 7.6; all Sigma-Aldrich, St. Louis, MO, USA) for 16 h at 56 °C, followed by Proteinase K inactivation at 100 °C for 10 min. To determine the amount of DNA, the cell lysates were treated with 0.415 IU/mL heparin and 1.25 µg/mL RNase for 30 min at 37 °C, followed by addition of 0.375 µL CYQUANT GR solution (ThermoFisher, Waltham, MA, USA). The samples were analysed using a SpectraMax Gemini plate reader with an excitation of 480 nm and an emission of 520 nm. As a standard, DNA sodium salt from calf thymus (Sigma-Aldrich, St. Louis, MO, USA) was used. To determine the amount of GAG, the cell lysates were diluted in PBS supplemented with 10 mM EDTA (pH 6.5) to a volume of 50µL and mixed with 200 µL of 32 mg/L 1,9-dimethylmethylene blue (DMB, Sigma-Aldrich, St. Louis, MO, USA) in 0.04 M Glycin, 0.04 M NaCl pH 3.0. Then the absorbance was measured on a Versamax microplate reader at 590 nm and 530 nm. A 530:590 nm ratio was used to determine the glycosaminoglycan concentration. As a standard, chondroitin sulphate sodium salt from shark cartilage (Sigma-Aldrich, St. Louis, MO, USA) was used.
5
Ethidium Bromide Binding to DNA
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