Alkaline Phosphatase Staining for Osteogenesis

Alkaline phosphatase staining was carried out as described previously.^{44 (link)} The cells were seeded in 6-well plates at a density of 2 × 10⁴ cells per well. After in vitro osteogenic induction for 7, 14, and 21 days performed as previously described, alkaline phosphatase staining was carried out. Briefly, culture wells were rinsed three times with PBS, fixed in 95% alcohol for 1 min, washed three times with PBS, incubated with the substrate mixture (2% sodium β-glycerophosphate, 25 ml; 2% sodium barbiturate, 25 ml; distilled water, 50 ml; 2% calcium chloride, 5 ml; 2% magnesium sulfate, 2 ml; and several drops of chloroform; Solarbio, Beijing, China) at 37 °C for 2 h, washed twice with pure water, incubated in 2% cobalt nitrate (Solarbio) for 2 min, and finally incubated with 2% amine sulfide (Solarbio) for 2 min. Stained cell images were captured at the same magnification and light intensity.