Alcian blue staining kit

BMCs were plated in triplicate in 4-well plate (SPL Life Sciences, Pocheon-si, Korea) for chondrogenic a pellet cultured incubated at 37 °C and 5% CO₂. After one day, the culture medium was removed, and then replaced with StemPro chondrogenesis (Thermo Fisher Scientific, Waltham, MA, USA) medium. Osteogenic and Adipogenic differentiation were incubated at 37 °C and 5% CO₂. After reaching 80% confluence, the culture medium was removed, and StemPro osteogenesis and StemPro adipogenesis (Thermo Fisher Scientific, Waltham, MA, USA) were added to the cultures. All differentiation medium was renewed every three days, and their differentiation potential was examined after 15 days of differentiation. Chondrogenic differentiation was examined by staining with Alcian Blue staining kit (Lifeline Cell Technology, Frederick, MD, USA) to identify sulfated proteoglycans deposits. Osteogenic differentiation was examined by staining with 2% Alizarin Red staining kit (Lifeline Cell Technology, Frederick, MD, USA) to identify calcium deposits. Adipogenic differentiation was examined by staining with Oil Red O staining (Sigma-Aldrich, St. Louis, MO, USA) to identify lipid droplets.

Glycosaminoglycan (GAG) staining was carried out using the Alcian Blue Staining Kit (Lifeline Cell Technology, Frederick, MD, USA) on ATDC5 cells that had been fixed with 4% paraformaldehyde in PBS.