MC3T3-E1 Cell Proliferation Assay

The MC3T3-E1 cells were seeded in 96-well plates at a density of 1.0 × 10⁵ cells/mL and cultured in αMEM containing 10% FBS and 1% penicillin-streptomycin. After 24 h of culture, NFPs, 100 µg/mL of ascorbic acid (Nacalai Tesque), and 5 mM of β-glycerophosphate (Calbiochem, LaJolla, CA, USA) were added to the culture for an additional 24 or 48 h. Cell proliferation was assayed using the alamarBlue^® reagent (Bio-Rad, Hercules, CA, USA). After 45 min of incubation with the diluted alamarBlue^® reagent (1:10 in DPBS), the fluorescence intensity was measured using PlateReader AF2200 (Eppendorf, Hamburg, Germany) at excitation/emission wavelengths of 535/590 nm [26 (link)]. The control group treated only with the dispersant was designated as 100%, and the cell proliferation was calculated relative to this control.