2.6. Alkaline Phosphatase (ALP) Activity

The alkaline phosphatase activity was evaluated by means of Alkaline Phosphate Assay Kit (Colorimetric) (Abcam, Cambridge, UK). This kit uses 50 µL of p-nitrophenyl phosphate (pNPP) 5 mM as a phosphatase substrate, which was added to 80 µL of each supernatant in duplicate and incubated for 1 h at room temperature in the dark. The reaction was stopped by adding 20 µL of stop solution in each well. The pNPP turned yellow when dephosphorylated by ALP, and the absorbance was read at 405 nm wavelength through a microplate reader (Multiskan GO, Thermo Scientific, Inc., Waltham, MA USA). The ALP activity (mU/mL/min) was calculated by following the manufacturer’s protocol.