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Biochemical analysis of GAG and DNA content

YS Yuanyuan Shi
XH Xiaoqing Hu
JC Jin Cheng
XZ Xin Zhang
FZ Fengyuan Zhao
WS Weili Shi
BR Bo Ren
HY Huilei Yu
PY Peng Yang
ZL Zong Li
QL Qiang Liu
ZL Zhenlong Liu
XD Xiaoning Duan
XF Xin Fu
JZ Jiying Zhang
JW Jianquan Wang
YA Yingfang Ao
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The wet weights of the OA cartilage explants were determined (n = 6 per group). After grinding, explants were digested overnight in papainase (125 μg ml−1) at 60 °C. The GAG content was measured using a DMMB assay. Lysates (20 μl) were mixed with 200 μl of DMMB working solution for 30 min at room temperature. The absorbance was then measured at 525 nm. Chondroitin sulfate (Sigma) was used as a standard. The DNA content was determined using the Hoechst 33258 assay (Beyotime Biotechnology, Beijing, China). Briefly, 20 μl of lysate was mixed with 200 μl of Hoechst 33258 working solution and incubated at 37 °C for 1 h. Absorbance was determined at 360 nm for excitation and at 460 nm for emission. Calf thymus DNA (Sigma) was used as the standard. The GAG or DNA content was shown as micrograms of GAG or DNA per milligram of wet weight.

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