sGAG content was determined by DMMB dye-binding assay21 (link). Briefly, MSC and chondrogenic spheroids were washed and digested in 500 μL solution of 0.1 mg mL−1 papain extraction reagent at 65 °C in water bath for 18 h. 20 μL of the digested samples was mixed with 200 μL DMMB solution and the absorbance was measured at 525 nm using a microplate reader (PowerWaveX, BioTek, Winooski, VT). Serially diluted solution of chondroitin 4 sulfate was prepared as the standard and the sGAG content was calculated according to the standard curve. The DNA content of same samples was also measured using the Quant-iT™ PicoGreen dsDNA Assay Kit (Molecular Probes Inc., Eugene, OR) according to the manufacturer’s instructions. Fluorescence intensity was determined by a SpectraMax multidetection microplate reader (Molecular Devices, Inc., Sunnyvale, CA) using a wavelength of 480 nm (excitation) and 520 nm (emission). sGAG content from each sample was normalized to dsDNA content.
Protocol detail
Quantifying sGAG in Chondrogenic Spheroids
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Assay
Bath
Chondrogenic
Chondroitin 4 sulfate
Devices
Dmmb
Dsdna
Fluorescence
Molecular probes
Papain
Picogreen
Corresponding Organization :
Other organizations : Pennsylvania State University
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Variable analysis
independent variables
- Digestion of MSC and chondrogenic spheroids in 0.1 mg mL^-1 papain extraction reagent at 65 °C for 18 h
- SGAG content determined by DMMB dye-binding assay
- DNA content measured using the Quant-iT™ PicoGreen dsDNA Assay Kit
- Serially diluted solution of chondroitin 4 sulfate as the standard for sGAG content measurement
Annotations
Based on most similar protocols
Use a water bath at 65 °C to digest samples in 0.1 mg/mL papain extraction reagent for 18 h (Input protocol, Protocol 1, Protocol 2, Protocol 4, Protocol 5).
Use a microplate reader to measure the absorbance of the DMMB-sGAG complex at 525 nm (Input protocol, Protocol 1, Protocol 2, Protocol 4, Protocol 5).
Prepare a standard curve using serially diluted chondroitin 4 sulfate to quantify the sGAG content (Input protocol, Protocol 1, Protocol 2, Protocol 4, Protocol 5).
Measure the DNA content of the same samples using the Quant-iT™ PicoGreen dsDNA Assay Kit and a SpectraMax multidetection microplate reader (Input protocol, Protocol 1, Protocol 2, Protocol 3).
Normalize the sGAG content to the DNA content of each sample (Input protocol, Protocol 1, Protocol 2, Protocol 3, Protocol 4).
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