*2.13. Osteocalcin Content*

Osteocalcin concentration was determined by the test method of the OCN ELISA kit (Takara Bio, Kusatsu, Japan) by following the process described by Bukhari et al. [42]. Initially, 100 μL of the cell culture solution was added to a 96-well plate coated with antibodies and incubated for 1 h at 37 ◦C. After removing unbound material with washing buffer (50 mM Tris, 200 mM NaCl, and 0.2% Tween 20), horseradish peroxidase (HRP) conjugated streptavidin was added to each well and incubated at 37 ◦C for 1 h to bind to the antibodies. After washing the wells five times, tetramethylbenzidine (TMB) solution was added to each well and incubated at room temperature for 20 min in the dark. HRP catalyzed the conversion of a chromogenic substrate (TMB) to a colored solution, with a color intensity proportional to the amount of protein present in the sample. After adding the stop solution, the absorbance of the sample in each well was measured at 450 nm. Results are presented as the percentage change in activity compared to the untreated control. The concentration of osteocalcin in the serum was measured using the same method as that used for cultured cell samples.
