2.6. Staining of extracellular calcium deposits (Alizarin Red S assay)

The Alizarin Red staining assay was performed on the 21st day of differentiation, on hDPSCs that had been seeded on 12-well plates at a density of 20,000 cells/well and treated with BMP-2 or BMP-2 peptide. The medium was removed and the cells were washed with 1× PBS and then fixed in 10% formalin solution (10% v/v formaldehyde, 0.4% w/v NaH₂PO₄.H₂O, 0.65% w/v Na₂HPO₄) at room temperature for 30 min. Afterwards, formalin was removed, the monolayers were washed twice with sterile distilled H₂O and incubated in 2% w/v Alizarin Red S solution at room temperature for 25–45 min. Then, the cells were washed multiple times with sterile distilled water to remove excess dye. The cells were observed with a Nikon ECLIPSE TS-100 inverted optical microscope (objective lenses: 10× and 40×, ocular lens: 10×) and photographs of the stained monolayers were taken at 100× and 400× magnification using a Nikon COOLPIX P5100 camera.