Alp activity fluorometric assay kit

A commercially available ALP activity fluorometric assay kit (BioVision) was used according to the manufacturer’s protocol. Briefly, the samples were homogenized through sonication in Tris-HCl buffer and centrifuged at 10,000 g for 10 min. Then 100 μL of the supernatant was mixed with 20 μL of 0.5 mM 4-methylumbelliferyl phosphate (MUP) substrate and incubated at 25°C in the dark for 30 min. Finally, 20 μL of stop solution was added to the mixture, and fluorescence was measured at Ex/Em of 360/440 nm. Samples of 0.1-0.5 nmol of MUP substrate with purified ALP enzyme were used to create a standard curve.

ALP activity of samples containing MSC was quantified as an early-stage osteogenic marker. A commercially available ALP activity fluorometric assay kit (BioVision) was used according to manufacturer instructions. Briefly, the samples were homogenized in ALP buffer and centrifuged at 10,000g for 10 min. A 100 μL of the supernatant was mixed with 20 μL of 0.5 mM 4-methylumbelliferyl phosphate (MUP) substrate and incubated at 25 °C in the dark for 30 min. Then a 20 µL of stop solution was added to the mixture, and the fluorescence was measured at Ex/Em of 360/440 nm. Standards of 0.1–0.5 nmol of MUP substrate along with purified ALP enzyme were used for calibration.