ALP Activity Assay

To determine alkaline phosphatase (ALP) activity, osteoblasts were incubated in different conditioned media in 6-well plates at a density of 1 × 10⁵ cells/well for 7 days. After cells were lyzed with 100 μL of assay lysis buffer, the ALP activity levels were determined with an ALP reagent kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) following the manufacturer’s instructions. Lysis concentration was adjusted for assays in a 96-well plate. ddH₂O, 5 μL, was added to the control well and 5 μL of phenol application liquid was used in the standard well. Together with control and standard well, lysis samples were supplemented with 50 μL of buffer and 50 μL of matrix liquid. The plate was then incubated at 37°C for 30 min, and 150 μL chromogenic agent was added to all wells and the plate gently mixed. Absorbance was read at 520 nm on a microplate reader and ALP activity was calculated.