The isolation of primary osteocytes was performed according to the published method (50). Primary osteocytes were isolated from mouse (4 months old) long bones according to the protocol (50). Long bones (femur and tibia) were aseptically dissected from skeletally mature mice and placed in α-MEM with 1% P/S. Following removal of epiphyses, bone marrow, and periosteum with scraping, extensive washing, and centrifugation, cortical bones were cut into 1 to 2 mm in length. Bone pieces were digested in collagenase solution [collagenase type IA (300 U/ml); Sigma-Aldrich] and dissolved in α-MEM for 25 min. The solution was aspirated and kept for cell plating, and the bone pieces were washed in Hanks’ balanced salt solution (HBSS). Subsequently, bone pieces were incubated with EDTA [5 mM (pH 7.4); Sigma-Aldrich] prepared in magnesium- and calcium-free Dulbecco’s PBS (Mediatech) with 1% bovine serum albumin (BSA; Sigma-Aldrich) for 25 min. The solution was aspirated, centrifuged (200g for 5 min), and kept for cell plating, and the bone pieces were washed again in HBSS. These steps were repeated for a total of three digestions. The remaining bone pieces were minced with a tissue homogenizer (Medimachine, BD Biosciences) in α-MEM, and the bone particles were directly plated. The digestions took place in a six-well petri dish on a rotating shaker set to 200 rpm at 37°C in a 5% CO2 humidified incubator. The collected cell suspensions were cultured on type I rat tail collagen-coated six-well plates at a seeding density of approximately 250,000 cells/9.5 cm2 in α-MEM supplemented with 10% FBS and 1% P/S. Cells were maintained at 37°C in a 5% CO2 humidified incubator for 7 days.

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