4.3. Alkaline Phosphatase (ALP) Enzyme Activity Assay

To compare the ALP enzymatic activity between live cells and ultrasonicated cell nanofragments, ATDC5 cells were cultured until confluency, trypsinized, harvested by centrifugation, and resuspended in basal culture medium. A total of 2 × 10⁶ cells were aliquoted in a 1.5 mL tube and centrifuged. The basal medium (supernatant) was then aspirated and replaced with 500 μL of Milli-Q ultrapure water. The cells were then submitted to ultrasonication for 30 s or 3 min on ice, and 500 μL of a p-nitrophenyl phosphate (pNPP) solution containing 1.0 mg/mL pNPP, 0.2 M trizma buffer and 5 mM magnesium chloride (SIGMAFAST™ tablets, Sigma-Aldrich) was added to the tube containing the freshly-prepared cell nanofragments.

In the case of live cells, after centrifugation, they were kept on ice without the addition of ultrapure water in order to avoid cell lysis due to the difference in osmotic pressure. The 1:1 diluted pNPP solution was added to the tube containing the live cells at the same time the pNPP solution was mixed with the freshly-prepared cell nanofragments.

After incubation for approximately 10 min at room temperature, 100 μL of the water-soluble yellowish hydrolyzed product of each sample was transferred into a 96-well plate, and 25 μL of 3 M sodium hydroxide solution was immediately added into each well to stop the reaction. The intensity of the colorimetric substrate was measured by a microplate reader (FlexStation 3 Multi-Mode Microplate Reader, San Jose, CA, USA) at the absorbance of 405 nm wavelength, available at the Central Research Laboratory, Okayama University Medical School. Experiments were performed in duplicate or triplicate samples.

The alkaline phosphatase enzyme activity in the live cells and in the cell nanofragments was estimated based on the concentration of the standard enzyme (5 U/μL, BioDynamics Laboratory Inc., Tokyo, Japan).