Alizarin red staining and quantification

After 21 days of culture in the different sera, osteogenic differentiation was evaluated by Alizarin Red staining to visualize calcium-rich deposits produced by the cells. The samples were washed twice in PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at 4°C, and stained with Alizarin Red solution (2%, pH 4.2; Sigma Aldrich, Milan, Italy) for 20 min at room temperature. Stained cells were extensively washed with deionized water to remove any nonspecific precipitation. Micrographs were taken with a microscope Eclipse TE2000-S (Nikon, Firenze, Italy) and a Nikon camera (Nikon, Firenze, Italy).

For the quantification of Alizarin Red S staining, 4 ml 10% (vol/vol) acetic acid was added to each well and the plate was incubated at room temperature for 30 min under gentle agitation. The monolayer was scraped off the plate and transferred to a 1.5 ml microcentrifuge tube after adding 1 ml of 10% (vol/vol) acetic acid. After vortexing for 30 s, the slurry was overlaid with 1.25 ml mineral oil (Sigma-Aldrich, St. Louis, MO), heated to 85°C for 10 min, and transferred to ice for 5 min. The slurry was then centrifuged at 20,000 g for 15 min, and 500 μl of the supernatant then removed to a new 1.5 ml microcentrifuge tube. 200 μl of 10% (vol/vol) ammonium hydroxide was added to neutralize the acid. Absorbance of aliquots (150 μl) of the supernatant was measured in triplicate at 405 nm in a 96-well format.