Cetylpyridinium chloride cpc solution

Cells were seeded at 1.5 × 10⁴ cells/well in 48-well plates and cultured in a complete medium for 24 h. Then, the old medium was replaced with the osteogenic/odontogenic medium with or without CBD (0, 0.1, 0.5, and 2.5 µM) and TNF-α (50 ng/mL), respectively. The medium was refreshed every 3 days. On day 14, after removing the old medium and washing it twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min. The cells were washed twice with PBS and stained by ARS staining (Solarbio, Beijing, China) for 5 min at room temperature. The microscope was used for observation and photography. For semiquantitative analysis, OD values of the solution at 562 nm wavelength were recorded after the mineralized nodules were completely eluted by 10% cetylpyridinium chloride (CPC) solution (Sigma-Aldrich, St Louis, MO, USA) for 30 min.

A total of 5 × 10⁴. HOBs/disk were cultured on the collagen disk into the 24-well culture plate, and after 14 days of culture, the cells were fixed with a glutaraldehyde solution (2.5%) for 2 h. The deposits of calcium were evaluated by adding the Alizarin Red staining solution (Sigma-Aldrich, St. Louis, MO, USA) to the culture for 1 h at room temperature. After eliminating the excess dye-deionized water, images were taken by a camera. The intensity of the red color showed the presence of calcium deposits. Next, 1 mL of 10% Cetylpyridinium Chloride (CPC) solution (Sigma-Aldrich, St. Louis, MO, USA) was added to the samples for 1h to chelate the calcium ions. This was used to quantify the calcium nodules. After 1h of incubation, the absorbance was read at 540 nm by a microplate reader (Synergy H1 Hybrid BioTek Instruments, Winooski, VT, USA).