Micromass culture and Alcian blue staining

C3H10T¹/₂ cells were plated in micromass as previously described (39, 70). When indicated, micromasses were cultured with BMP2 (100 ng/ml; R&D) or 7 μM verteporfin (Sigma-Aldrich) or vehicle. After 3 days, micromasses were harvested for real-time PCR gene expression analysis or fixed and whole mount stained with Alcian blue. Images were acquired at room temperature with a Leica DFC295 camera on a dissecting microscope. Alcian blue staining, extraction, and quantification were performed as previously described (49, 70). Densitometry of the micromasses was measured using ImageJ (71), by placing all images on a single canvas, splitting into hue, saturation, and brightness stacks, and analyzing the “saturation” stack with the gel tool. The entire micromass was selected for analysis.