Bone resorption assays.
1) Use dentin slice as bone chip.
2) Place dentin slices in 70% EtOH, RT for overnight.
3) Take out the dentin slices from the tube containing 70% EtOH, then put them into
medium with 10% serum or total serum, 37°C for overnight.
4) Put the dentin slices into culture wells and seeded osteoclast precursors (MBMC 1×
105/well, or HBMC 1×105/well, or RAW 5000/well), added indicated treatments. Half of
the media with the same treatment refreshment were changed every 2-3 days, 37°C for
indicated times (MBMC 7days, HBMC 21days, RAW 4-5days).
5) Osteoclast formation was observed by TRAP staining (using TRAP staining Kit, Sigma)
and bone resorption
6)
lacunae were stained with hematoxylin.
The cells on the dentin slices were removed by rubbing the slices, and the number
of resorption pits was scored microscopically.