4.7. Alkaline Phosphatase (ALP) Assay

The ALP assay was performed for the measurement of cells’ ALP as an indication for early osteogenesis. Briefly, at Days 2, 7, 14, 35, and 63, samples, i.e., PCL/CaP scaffolds cultured with OB or OB+PBMC, were transferred to a new 48-well plate, washed twice with PBS, and incubated in 250 µL of an ALP reaction solution (3.5 mM 4-nitrophenyl phosphate di-sodium salt hexahydrate in an ALP buffer) for 30 min at 37 °C. A reaction solution without cells was used as a control. At the same time, an AP standard solution was prepared by serial dilutions of a 1 mM 4-nitrophenol solution in an ALP buffer (50 mM Glycine, 100 mM Tris-Base and 2 mM MgCl₂) at pH 10.5. After incubation, 100 µL of supernatant diluted at a 1:1 ratio in PBS or a standard solution was transferred to a 96-well plate in triplicate. Finally, absorbance was measured at 405 nm using a Fluostar Omega Plate Reader. ALP measured was normalized with dsDNA content obtained from a PicoGreen assay.