Axiovert 25 inverted phase microscope

Chondrogenesis was induced by using the StemPro Chondrogenesis Differentiation Kit (A1007101, Thermofisher). Briefly, cells were seeded in micromasses (80,000 cells/each) and incubated for 2 h in a humidified chamber in the incubator before differentiation medium was added. After 28 days, the chondrogenic micromasses were either harvested into Trizol for gene expression measurements or fixed in paraformaldehyde (4%) for 45 min and directly stained overnight with Alcian Blue (1%; Sigma). Pellets were imaged in a Zeiss Axiovert 25 Inverted Phase microscope and radius measured with Zen 2 to calculate the spherical area (A = 4πr², where A is the area and r the radius).

To induce adipogenesis cells were seeded on collagen (0.2%) in 24-well plates (50,000 cells/well) and grown until confluence as previously described (5 (link)). Cells were kept for 5 days in differentiation medium consisting of DMEM supplemented with 7% Rabbit Serum (Gibco), 3% FBS, 1% P/S, 1 μM dexamethasone (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich) and 0.5 mM 3-isobutyl-1-methylxanthine. This was followed by 7 days of cell culture in DMEM with 10% FBS, 1% P/S and 10 μg/ml insulin. Medium was changed every 2–3 days. In order to visualize lipid accumulation, differentiated adipocytes were stained with Oil red O (0.375% prepared in isopropanol) for 10 min and imaged in a Zeiss Axiovert 25 Inverted Phase microscope, and differences between the two types of cells were assessed visually.