α mem complete medium

Bone marrow cells were obtained from the femur and tibiae of the C57BL/6 mice by flushing with Dulbecco's modified Eagle's medium [DMEM (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), sodium bicarbonate (2.2 g/ml) using an intradermal needle (BD precision glide). Cells were suspended in the same medium supplemented with penicillin (1664 U/ml) and streptomycin (745 U/ml).
BMMC were separated with Histopaque 1083 (Sigma—Aldrich, Saint Louis, CA) and re-suspended in α-MEM complete medium (Sigma—Aldrich) [supplemented with 15% fetal calf serum (FCS), sodium bicarbonate (2.2 g/ml), penicillin (100 U/ml), streptomycin (100 U/ml), and gentamicin (Sigma-Aldrich), L-Glutamin (200 mM) (Sigma-Aldrich), MEM non-essential amino acids 1% (Gibco)]. α-MEM complete medium was added with 20 ng/ml macrophage-colony-stimulating factor (M-CSF, (Peprotech, Rocky Hill, NJ) which promotes survival and proliferation of osteoclast precursors (Ross and Teitelbaum, 2005 (link)).

BMMC (2 × 10⁵) were cultured in 96-well plates (Corning—Costar) in α-MEM complete medium (Sigma—Aldrich) supplemented with 20 ng/ml M-CSF and with or without 50 ng/ml RANKL (Peprotech, Rocky Hill, NJ) (Axmann et al., 2009 (link); Makihira et al., 2011 (link)). AaCDT was added to each well in concentrations of 0, 12.5, and 25 μg/ml. The cells were incubated at 37°C with 5% CO₂ in a fully humidified atmosphere for 6 days and maintenance was done every 2 days by removing 50% of the culture medium and adding the same volume of medium and its supplements.
Negative control cells were cultured in medium containing RANKL (50 ng/ml) and 100 ng/ml osteoprotegerin (OPG, Peprotech, Rocky Hill, NJ), whereas positive control cells were cultured in medium containing an optimal concentration of RANKL (100 ng/ml). After 6 days of culture, cell viability, the number of TRAP—positive multinuclear cells and gene expression were determined.