Isolation and Culture of Primary Osteocytes

Primary osteocytes were isolated from marrow-flashed long bones of 18-month-old trans genic mice and their WT littermates, as previously described [23 (link)]. Briefly, long bones were dissected and sequentially digested with digestion solution containing 0.15% (v/v) collagenase type I (BD Biosciences, Concord, MA, USA) and EDTA (5 mM) in an incubator under 37 °C and 5% CO₂. The cells isolated from the first three digestions were removed and the final digests enriched osteocytes were cultured in alpha Modified Eagle’s Medium (α-MEM, Gibco, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Shanghai, China). After 48 h, the CM was collected and stored at −80 °C until use. The C2C12 myoblasts were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Carlsbad, CA, USA) containing 10% (ν/ν) fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Beyotime, Haimen, China) for 48 h. All cell lines were cultured under 5% CO₂ and 37 °C in a controlled humidified incubator.