Recombinant murine rank ligand rankl

Cytotoxicity of sealer extracts in BMMs was evaluated at 12 h, 24 h and 48 h. MTT solution (Sigma-Aldrich) (5 mg/mL) was added to each well, and the cells were incubated at 37ºC for 4 h. Supernatants were removed, and 100 μL of dimethyl sulfoxide (LGC Biotecnologia) was added. The optical density at 570 nm was measured using a microplate reader (Biochrom, Cambridge, EN, United Kingdom).
Differentiation of BMMs into osteoclasts (osteoclastogenesis), exposure to the sealer extracts, and tartrate-resistant acid phosphatase (TRAP) stain BMMs were exposed simultaneously to the sealer extracts, 50 ng/mL of recombinant murine RANK Ligand (RANKL) (PeproTech), and 30 ng/mL of M-CSF (PeproTech) during a 5-day period. The extracts and reagents were replaced on day 3. The positive control group contained cells kept in 10% α-MEM stimulated with M-CSF and RANKL, and the negative control group contained cells maintained only in 10% α-MEM and M-CSF (not induced to undergo differentiation). At day 5, the cells were stained for TRAP using a commercially available staining kit (Sigma-Aldrich), according to the manufacturer's protocol. TRAP- positive multinucleated (> 3 nuclei) cells were counted as osteoclasts, and expressed as a percentage. Images were obtained at 10x magnification using a Leica DM IRB-inverted microscope coupled to a DFC490 camera (Leica, Wetzlar, HE, Germany).