Diethanolamine

Plasma samples (10 μL) were brought to 0.2 mL with the following reaction conditions: 0.2 M Diethanolamine and 1 mM MgCl (Merck Life Science S.L.U, Madrid, Spain), pH 9.8, incubated with 4 mM p-nitrophenyl phosphate (Merck Life Science S.L.U, Madrid, Spain) at 37 °C with agitation. All samples were assayed in duplicate. The background signal was determined by pre-incubating plasma samples with 150 μM levamisole (a TNAP-specific inhibitor) for 5 min, 37 °C, before adding 4 mM p-nitrophenyl phosphate in the presence of levamisole. The absorbance of the samples was then measured at 405 nm, 25 °C, in a spectrophotometer.

After 14 days of incubation, samples were washed with phosphate-buffered saline (PBS) and then frozen at −80 °C until analysis was conducted.
To investigate cell proliferation and osteogenic differentiation, determination of DNA content (proliferation) and alkaline phosphatase (ALP, osteogenic differentiation) activity was carried out. For this purpose, frozen samples were thawed and lysed with 1% Triton X-100/PBS (Merck, Darmstadt, Germany) for 50 min on ice. Cell numbers were then determined using the QuantiFluor^® dsDNA system (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Fluorescence was quantified using a microplate reader (infinite M200 PRO, Tecan, Switzerland) and correlated to a calibration line generated from defined numbers of cells from the same experiment.
To measure ALP activity, cell lysates were incubated with 1 mg/mL p-nitrophenylphosphate in 0.1 M diethanolamine (pH 9.8) containing 1% Triton X-100 and 1 mM MgCl₂ at 37 °C for 30 min (all from Merck). After stopping the enzymatic reaction by adding 1 M NaOH (Merck), absorbance was measured at 405 nm. Values were correlated to a calibration line generated from different dilutions of a 1 mM p-nitrophenol stock solution (Merck) and normalized to the cell number.