Bovine calf serum (bcs)

Primary osteocytes were isolated from long bone diaphyses (femur, tibia, and humerus) from five mice of each age and sex according to published methods (Stern et al., 2012). Briefly, long bones (two femurs, two humerii, and one tibia per mouse) were harvested aseptically, after which epiphyses were removed and marrow was flushed from the diaphysis. The remaining tibia from each mouse was fixed in 10% neutral buffered formalin for histology. The flushed cortical bone shafts from all mice in each group were collected and pooled, then separated into three separate batches (n = 8 to 9 diaphyses each) for cell line isolation. Bone shafts were subjected to serial digestions with collagenase (Type 1A, Sigma‐Aldrich #C9891) and EDTA, and cells from digests 7 through 9 along with cells from bone chip outgrowth were pooled and considered to be an osteocytic population, as described (Stern et al., 2012). Cells were plated onto type 1 collagen‐coated dishes in osteocyte culture medium [α‐MEM (Invitrogen) + 5% fetal bovine serum (FBS, Atlanta Biologicals) + 5% bovine calf serum (HyClone) + 1% antibiotic/antimycotic)] and grown to 70% confluency, at which time cells were re‐seeded for all subsequent experiments. All primary osteocyte experiments were conducted within 2 weeks of isolation, as recommended (Stern et al., 2012).