DPCs 5 104 cells were seeded into a 6-well plate before cultivation in snail mucus
supplemented DMEM (2 ml/well) and maintained at 37°C in 5% CO2 for 3 weeks. Culture
medium was changed every 3 days. Mineralized nodules were visualized by staining with
Alizarin red staining. Briefly, the cells were fixed with 4% formaldehyde for 5-10 min and
stained with 0.5% Alizarin Red S pH 4.2 for 5 min. The cells were then rinsed with tap water
and viewed with an inverted microscope (Nikon ECLIPSE TS 100). Mineralized nodules were
counted and reported as number of nodules per well compared to that of the control group
which was cultured in complete DMEM. Three replicate assays were performed for the
statistical analysis.