4.6. Alkaline Phosphatase(ALP) and DNA Quantification

To quantify ALP catalytic activity in the 3D Osteogel differentiation, an ALP quantitative assay using 4-nitrophenyl phosphate (pNPP) as a substrate was used as previously described [29,52,53,54]. Briefly, differentiated cells in Osteogel were rinsed with DPBS, collected in 1.5 M Tris-HCl lysis buffer (1 mM ZnCl₂, 1 mM MgCl₂ and 1% Triton X-100) pH 10, and homogenized by sonication. The supernatant atop lysates was incubated with 100 μL pNPP substrate solution (3.7 mM pNPP in 100 mM diethanolamine and 0.1% Triton X-100, pH 9.8) at 37 °C for 30 min. Absorbance of the released 4-nitrophenolate was measured by a spectrophotometer at 405 nm wavelength. The overall concentration of ALP was calculated against the values of the p-nitrophenolate standard curve.

To normalize the ALP activity to the viable cell number inside the Osteogel, the DNA content was quantified using Quant-iT™ PicoGreen™ dsDNA Assay (ThermoFisher Scientific Inc., Waltham, MA, USA). Briefly, the Osteogel lysates from the previous step were added to a flat-bottom 96-well plate containing PicoGreen solution and incubated in the dark for 5 min. The fluorescence of PicoGreen was measured by a spectrophotometer set at 532 nm wavelength. The DNA concentration was calculated from the standard curve derived from the dilution of Lambda DNA standard (100 μg/mL) provided by the kit. In all samples, the average ALP was calculated from the overall ALP normalized to the DNA content of the samples, as the ALP activity per 1 μg DNA.