Osteoblast alkaline phosphatase (ALP) activity

ALP activity of human cultured osteoblasts was examined using an ALP activity detection kit (cat. no. P0321; Beyotime Institute of Biotechnology, Inc.). Following stimulation by 10 ng/ml of transforming growth factor (TGF)-β1 and 4 mM L-glutamine for 3 days, osteoblasts were treated with miRNA or miRNA antagonist for 2 days following pre-induction with TGF-β1 and L-glutamine. ALP activity was determined in the cell culture supernatants and osteoblasts lysates, according to the manufacturer's protocol. All standards, test samples and buffers were pre-warmed to room temperature. A total of 50 µl/well of p-nitrophenol as a substrate was added to a 96-well plate and 100 µl of standard or unknown samples were mixed well with the substrate. The 96-well plate was incubated at 37˚C in an incubator for 10 min and the reaction was stopped by adding 100 µl of stop solution. The plate was read using a plate reader at a wavelength of 405 nm (BioTek Synergy 2; BioTek Instruments). ALP activity of the unknown samples was calculated according to the standard sample activity divided by the reaction time of 10 min.