Human Osteoblasts-femural (HO-f)

In order to test the ALP activity (osteogenic differentiation), cells were harvested using collagenase I and trypsin, centrifuged at 1500 rpm for 5 min, resuspended in 1 ml of cold PBS (4 °C) and counted. A volume corresponding to 300,000 cells was taken, centrifuged and suspended in 600 μl of assay buffer, and centrifuged at 13,200 rpm for 5 min at 4 °C. The supernatant was transferred to a new tube. Then 200 μl of the supernatant was distributed in four wells of a 96-well plate (50 μl/well). One of these wells served to determine the background through the simultaneous addition of 80 μl p-nitrophenyl phosphate (pNPP) 1 mM and 20 μl of stop solution. For the three remaining wells, 80 μl of pNPP was added. After 60 min, 20 μl of stop solution was added to the wells and the plate was read in a spectrophotometer to measure the optical density at 405 nm. The quantity of p-nitrophenol (pNP) in each well was determined using a standard curve established using pNPP and purified ALP enzyme. Then 350 μl of the remaining supernatant was incubated with 750 nM of Hoechst 33342 for 30 min and distributed in triplicate in an opaque 96-well plate to read the fluorescence at 365 nm excitation.