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4 protocols using mlo y4

1

Effects of Dexamethasone and Hypoxia on Osteocyte and Mesenchymal Stem Cell Viability

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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.
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2

Osteocyte-like Cell Culture Protocol

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Murine Osteocyte-like cell line MLO-Y4 (Kerafast, Inc. Boston, MA) were cultured according to the suppliers recommended protocol.[76 (link)] The cell culture flasks were coated with 4μg/cm2 rat tail type I collagen (Sigma-Aldrich, Inc. St. Louis, MO) for 30 minutes at 37°C before cell seeding. Alpha-Minimum Essential Media (Thermo-Fisher Scientific, Grand Island, NY) supplemented with 2.5% fetal bovine serum (FBS, R&D Systems, Minneapolis, MN), 2.5% calf serum (Cytiva Life Sciences, Marlborough, MA) and 1% penicillin/streptomycin (Thermo-Fisher Scientific) were used as cell culture media. The cells were seeded on collagen coated glass slides (Fisherbrand, Thermo-Fisher Scientific) at a density of 10,000 cells/cm2 and cultured for a period of 1 to 3 days. Human osteoblast like Saos2 osteosarcoma cells (ATCC, Manassas, VA) were cultured with Dulbecco’s modification of eagle’s media (DMEM, Thermo-Fisher Scientific supplemented with 10% FBS (R&D Systems), 1%penicillin/streptomycin (Thermo-Fisher Scientific) and 1% Glutamax (Thermo-Fisher Scientific) using standard cell culture protocol.
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3

Murine Osteocytes Under Hypoxia and Dexamethasone

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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.
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4

Hypoxia-Induced Osteocyte Cell Culture

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MLO-Y4 (kerafast, Boston, USA) murine cultured osteocytic cells, which have been used previously, were cultured 16 (link). The cells were plated on type I collagen-coated dishes (BD Biosciences, Bedford, USA) and cultured in α-minimal essential medium (α-MEM) supplemented with 2.5% (v/v) FBS, 2.5% (v/v) FCS, streptomycin (100 μg/ml) and penicillin(100units/ml) 17 (link). Then for the hypoxia experiments, the cells were incubated for 24h in a CO2/tri-gas incubator (Astec, Fukuoka, Japan) set at a mixture of 5% (v/v) CO2 and 1% (v/v) O2 balanced with N25 (link).
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