Isolation and Culture of Murine Osteocytes

Osteocyte-enriched fractions derived from mouse long bones were isolated as previously reported by Stern et al. [51 (link)]. Cell suspensions from individual mice were cultured in twelve-well plates coated with type-I rat tail collagen (Millipore Sigma) at a seeding density of 1 × 10⁵ cells per well. In α-minimal essential medium supplemented with 2.5% fetal bovine serum (FBS), 2.5% bovine calf serum (BCS) and 1% penicillin and streptomycin (PS) (Thermo Fisher, Hampton, NH, USA). Cells were maintained at 37 °C and 5% CO₂ in a humidified incubator. Osteocytes were not disturbed for the first 7 days while they attached to the plate. Media changes were performed every 48 h beginning on day 7, and conditioned media was collected from osteocyte cultures derived from individual mice every 48 h on days 9, 11, 13, and 15 post isolation. Conditioned media (CM) was filtered using a 0.22 µm syringe filter and aliquoted before storing at −20 °C. Serum-free CM was collected for mass spectrometry and immunodepletion assays after a 4 h incubation on day 7 of cultures.