ALP assay
Next, 5 × 104 MC3T3-E1 cells/mL in α-MEM were seeded in 6-well plates overnight. The medium in each well was removed the next day and replaced with osteoblast differentiation-inducing medium (OIM) (MK430, Takara Bio Inc., Tokyo, Japan), and then incubated at 37 °C for 3 weeks in a 5% CO2 incubator. Day 0 is the date the OIM medium was added. ALP activity was determined using an ALP assay kit (ab83369, Abcam, Cambridge, MA, USA) following the manufacturer’s instructions. In brief, untreated control and NaF-treated MC3T3-E1 cells were homogenized in assay buffer after washing with cold PBS and centrifuged at 15,000 rpm for 15 min at 4 °C. For each measurement, the supernatants were added in triplicate in 96-well plates and each well was brought to a total volume of 80 µL with assay buffer, followed by the addition of 20 µL stop-solution for a background control. Standard curves were generated as detailed in the manufacturer’s manual. The ALP activity of each sample was calculated based on a comparison between the standard and sample curves and is reported as Unit/mL (U/mL).