Alkaline phosphatase staining

Mouse bone marrow-derived MSCs were developed as reported. Mouse MSC and C3H10T1/2 cells were seeded in six-well plates and cultured for 7 days in osteogenic differentiation medium with or without azoramide. The cells were then rinsed three times with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature for 15 min, and washed three more times with PBS. For staining, an alkaline phosphatase (ALP) substrate solution (Beyotime, Shanghai, China) was added to the fixed cells for 30 min at room temperature. Cells were then washed three times with distilled water, and images were photographed.