Quantitative Assessment of Osteoblastic Differentiation

Alkaline Phosphatase (ALP) activity is a biochemical marker for osteoblast activity. Briefly, ALP production by the DPS-7 cells was assessed on days 7 and 14 of cell treatment with probiotic CM. First, 10⁵ hDPSC were cultured per well of a 24-well plate. After 7 and 14 days, cells were washed 3 times using Phosphate-Buffered Saline (PBS) solution. Cells were homogenized in 0.5 ml of RIPA buffer (pH=7.5, 0.05% Triton X-100, 1 mM MgCl₂, 10 mM Tris-HCl) using sonication and then centrifuged at 12,000 rpm for 10 min at 4°C. Cell degradation contents (0.1 ml of the floating contents) were mixed with 0.5 ml of p-Nitrophenol Phosphate (PNP) (Sigma-Aldrich, USA) solution and 0.5 ml of alkaline buffer solution (Sigma-Aldrich, USA). After incubation at 37°C for 15 min, 1 ml of 0.02 N NaOH was added to the mixture to stop the interaction and adsorption was measured at 405 nm using a spectrophotometer (Bio-Rad, USA) ^{8 (link)}. A standard curve of known PNP concentrations was plotted and used to calculate the concentration of the samples. The ALP activity was calculated as the ratio of the PNP nanomoles converted per min to milligrams of the total protein. Total protein was measured using the Bradford assay.