Activity of ALP in MSC was measured after exposure to the different experimental conditions for 7 days. After washing with PBS, cells were lysed with 400 µl ALP lysis buffer (150 mM Tris pH 10.0, 0.1 mM ZnCl2, 0.1 mM MgCl2, 1% Triton-X100) at room temperature under constant agitation for 30 min. Supernatants were immediately frozen at − 80 °C. For measurement of ALP activity, an aliquot was centrifuged for 10 min at 12,000 rpm and 4 °C. Each sample was measured in triplicate. 50 µl per well were mixed with 200 µl pre-warmed (37 °C) substrate solution (ALP buffer with freshly dissolved p-Nitrophenyl phosphate at 2.7 mM) in 96-well plates. Optical densities (OD) were measured at 405 nm and followed over a 60 min incubation period at 37 °C in intervals of 10 min. A time point during the linear phase was chosen and ∆OD values to baseline ODs were calculated and divided by the protein concentration of the sample as determined with the DC Protein Assay (Bio-Rad) for normalization. Finally, each ∆OD/protein ratio was related to the ∆OD/protein ratio of the appropriate control.

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