Mlo y4

An established murine osteocyte cell line (MLO-Y4) (Kerafast, Boston, MA) was maintained as a subconfluent monolayer culture in the MEM alpha medium (Gibco, Tokyo, Japan) supplemented with 10% fetal calf serum 10 (link). Mouse MSC derived from the bone marrow (Cyagen, Silicon Valley, CA) was maintained in the stem cell growth medium (Cyagen). Both cell lines were cultured at 37°C under 20% O₂ and 5% CO₂. At the culture reached 80% confluency while being cultured at 37°C under 20% O₂ and 5% CO₂, both cell lines, MLO-Y4 and MSC, were treated under three different conditions for 24 hours: exposed to Dexamethasone (Dex, MSD, Tokyo, Japan) at the concentration of 0.4 ng/ml (Dex group); hypoxia at a 1% oxygen concentration (Hypoxia group) or both (Dex/Hypoxia group). As a control (C group), both cell lines were cultured under 20% oxygen in the culture medium without Dexamethasone. After the exposure, the numbers of viable and nonviable cells determined using 0.25% trypan blue dye exclusion method were counted with Countess 2 FL (Thermo Fisher Scientific, Waltham, MA), and survival rates were calculated.

An established murine osteocytic cell line (MLO-Y4) (Kerafast, Boston, MA, USA) was maintained as a subconfluent monolayer culture in the alpha MEM medium (Gibco, Tokyo, Japan) supplemented with 10% fetal calf serum. When the culture reached 70% confluency being cultured at 37 °C under 20% O₂ and 5% CO₂, MLO-Y4 were treated with 1 µM Dexamethasone (Dex, MSD, Tokyo, Japan) in 1% O₂ (hypoxia) for 24 h (Dex+/hypoxia+ group). A quantity of 0.8 µM taurine was added to the medium of Dex+/hypoxia+ group exposed to taurine-free medium (Dex+/hypoxia+/taurine+ group). As a control group, cells were cultured under 20% O₂ in the culture medium without either Dex or taurine (Dex-/hypoxia− group). Three independent experiments each were carried out.