Pnpp liquid substrate system

After culturing for 7 and 14 days in various conditions, the cells were assayed for ALP activity with a p-Nitrophenyl Phosphate (pNPP) Liquid Substrate System (Sigma) following the manufacturer’s instructions. For morphological observation, the cells were stained using an alkaline dye mixture of 0.01% (W/V) Naphthol AS-MX Phosphate Alkaline Solution and 0.24 mg/ml Fast Blue RR salt at 18–26°C for 30 min. After 30 min Mayer’s Hematoxylin Solution was used for staining for 10 min. For quantitative determination, cells were first lysed in lysis buffer (Sangon) for 30 min and then transferred to a 96-well transparent plate. Next, pNPP solution was added to each well, and the plate was incubated in the dark for 30 min at 37°C. The plate was read at 405 nm on a multifunction microplate scanner (Infinite M200, TECAN).