4 well plate

Protein stability was analyzed by incubating 1 ml screw-capped vials in deionized water, at 25 °C for 4 h to achieve complete equilibrium. The 1 ml samples were treated in a 4-well plate (SPL Life Sciences Co.) at 6 mm distances from the end of the quartz tube of the DBD plasma jet for different treatment times, and then incubated for 4 h at room temperature. The concentration of proteins after the plasma treatment is determined using the Bradford method46 (link). Three samples were treated for each condition to minimize the error.

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.