The cells after reaching 80% confluence were trypsinized, washed, and resuspended in high-glucose DMEM with 1 mM sodium pyruvate (Invitrogen), 0.1 μM dexamethasone (Sigma-Aldrich), 200 μM ascorbic acid (Sigma-Aldrich), 1 × insulin-transferrin-selenium (Invitrogen) and 10 ng/ml transforming growth factor-1 (Peprotech, London, UK). The viable cells were seeded in 15-ml conical tubes at a density of 5 × 105 cells per pellet. Next, the cells were gently allowed to centrifuge to the bottom of the tubes to form compact cell pellets, and then incubated in a humidified atmosphere in 5% CO2 at 37 °C. The medium should be changed every 3 days. R-2HG at a concentration of 0–1.5 mM was used for treatment from day 1.
Sections of paraffin-embedded MSCs pellets were processed for immunohistochemistry using rabbit anti-human collagen type II (Abcam, Cambridge, MA, USA). EnVision detection kit (Dako, Carpinteria, CA) was applied to analyze the immunoreactivity of the sections. Non-immune rabbit- IgG antibody was used as the negative control.
Sections of paraffin-embedded MSCs pellets were processed for immunohistochemistry using rabbit anti-human collagen type II (Abcam, Cambridge, MA, USA). EnVision detection kit (Dako, Carpinteria, CA) was applied to analyze the immunoreactivity of the sections. Non-immune rabbit- IgG antibody was used as the negative control.