Alizarin red and ALP staining

Alizarin red and ALP staining was used to analyze mineralized nodule formation and alkaline phosphatase activity of differentiated CON-ASCs and DOP-ASCs. CON-ASCs and DOP-ASCs were seeded on 12-well plates at 5 × 10⁴ cells per well. The medium was changed to osteogenic induction medium (Cyagen, Guangzhou, China), after CON-ASCs and DOP-ASCs were transfected with Si-Jkamp and plasmid (Jkamp), respectively. The medium was changed every 3 days. After 3 and 5 days of osteoinduction, the osteogenesis induction medium was discarded. Cells were washed twice with PBS and then fixed with 4% neutral buffered formalin for 30 min. ALP activity was detected using an Alkaline Phosphatase Assay Kit (Beyotime, Shanghai, China) in accordance to the manufacturer’s protocol. At 14 days after osteogenesis induction, we performed alizarin red staining (Cyagen) in accordance with the manufacturer’s protocol to assess the formation of calcium nodules. Staining was observed using the DFC 7000T system (Leica, Wetzlar, Germany). Each image was selected to save by a visual camera.