Soluble rankl srankl

For the osteoclastogenic culture, RAW264.7 and pNFAT/Luc-RAW cells were cultured in α-MEM medium containing 10% fetal bovine serum (FBS), 100 ng/ml soluble RANKL (sRANKL, Peprotech and Oriental Yeast), 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin [10] (link). For osteoclast formation by using primary pre-osteoclast cultures, murine bone marrow cells were obtained from femurs and tibiae of 7-week-old ddY mice (Japan SLC, Inc.). 4.0×10⁵ cells were cultured in α-MEM medium containing 10% fetal bovine serum (FBS), 100 ng/ml sRANKL, 10 ng/ml M-CSF (Wako, JPN), 2 mM l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in 24-well plates. Kenpaullone (Sigma), olomoucine (Sigma), roscovitine (Sigma) are used at a final concentration of 50 nM. The culture medium in each well was replaced by fresh medium containing M-CSF and sRANKL every 2 days. TRAP staining was performed after 5 days of the induction.

PBMCs isolated by density gradient centrifugation were plated in 96-well culture plates as described before.19 (link) PBMCs were left overnight for osteoclast precursors (OCPs) to adhere on bone slices (Immunodiagnostic Systems, Boldon, UK).20 (link) On the following day (day 1 of culture), medium was changed to Dulbecco's modified Eagle's Medium (DMEM) supplemented with macrophage colony stimulating factors (M-CSF) 25 ng/mL (Peprotech, London, UK). Three days later, medium was again changed to DMEM with M-CSF (25 ng/mL), soluble RANKL (sRANKL) (50 ng/mL; Peprotech), dexamethasone (10 nM; Sigma-Aldrich) and transforming growth factor β (2.5 ng/mL; R&D Systems) in order to differentiate the OCPs into mature OC.21 (link) The culture medium was then changed twice a week. Cells cultured on bone slices for 7, 14 and 21 days20 22 (link) were used for functional assays and gene expression.