For Col quantification on day 14, samples from cell groups were digested in 0.1 mg/mL pepsin (Sigma-Aldrich) in 0.2 M acetic acid at 37 °C for 16 hours. A Sircol assay kit (Biocolor Life Science Assays) was then used to quantify Col. For GAG and DNA quantification on day 14, other samples from cell groupings were digested in 125 μg/ml papain (Sigma-Aldrich) in 100 mM sodium phosphate buffer (pH 6.5) containing 10 mM L-cysteine and 10 mM EDTA at 65 °C for 16 hours. A Blyscan assay kit (Biocolor Life Science Assays) was used for GAG, and the Quant-iT™ PicoGreen™ dsDNA Assay Kit for DNA. The three kits were used following manufacturer directions as has been done in similar studies by others (Cai et al., 2015 (link); Coppola et al., 2018 (link); A. Nazempour et al., 2017 (link)).
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Quantification of Collagen, GAG, and DNA in Cell Cultures
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Corresponding Organization :
Other organizations : Washington State University, The University of Texas at San Antonio, Morehouse College, Anderson Orthopaedic Clinic
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Variable analysis
independent variables
- Cell groups
- Col quantification
- GAG quantification
- DNA quantification
- Pepsin concentration (0.1 mg/mL)
- Acetic acid concentration (0.2 M)
- Digestion temperature (37 °C)
- Digestion duration (16 hours)
- Papain concentration (125 μg/ml)
- Sodium phosphate buffer (100 mM, pH 6.5)
- L-cysteine concentration (10 mM)
- EDTA concentration (10 mM)
- Digestion temperature (65 °C)
- Digestion duration (16 hours)
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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