Stempro chondrogenic differentiation medium

Cells were diluted to a concentration of 4 x 106 cells/mL and three 50-μL drops of the concentrated cell suspension were placed in each well of a 6-well plate (Corning Life Sciences). The plate was incubated overnight at 37°C/5% CO2. After 24 h, chondrocyte differentiation medium (StemPro Chondrogenic Differentiation Medium, Gibco) was added to experimental wells, and culture medium was added to control wells and incubated at 37°C/5% CO2. Every three days, the media in each well was replaced in full. After a minimum of 2 weeks of incubation, media was aspirated from the wells, the cells were washed with DPBS, and 1 mL of 10% formalin (ThermoFisher, Carlsbad, CA, USA) was added to fix the cells overnight at room temperature. The formalin was aspirated and the wells were washed with deionized (DI) water, then 1 mL of Alcian blue was added to each well and incubated at room temperature for 45 min with gentle rocking. The dye was aspirated from the wells, a destain solution (2:3 [v:v] acetic acid to pure ethanol) was placed in the wells, incubated at room temperature for 10 min, and aspirated. After a second destaining, the destain was aspirated, and after drying at room temperature, the wells were imaged with a phase contrast microscope fitted with an Amscope MD camera.