ALP activity

Osteoblast specific ALP activity was measured using a colorimetric Alkaline Phosphatase Assay Kit (abcam, Milton, England) according to the manufacturer’s instructions. Upon termination of the experiment MG-63 cells were detached from the cell culture plate, collected at 1000 × g and 4 °C for 5 min and resuspended in 230 μl (24 h) or 350 μl (48 h) of ALP assay buffer. Lysis was performed by five repeated freeze/thaw cycles in liquid nitrogen at −196 °C and a water bath at 37 °C respectively. Cell lysates were stored at −20 °C until use. Prior to measuring the ALP activity of the cell lysates insoluble material was removed by centrifugation at 13.000 × g and 4 °C for 3 min. Aliquots of 80 μl of the supernatants were subjected to analysis in duplicate. The conversion of para-nitrophenylphosphate (pNPP) chromogenic substrate over 60 min was compared to a standard curve as specified by the manufacturer.