C - Michigan Technological University

5.2 Methods5.2.1 Black bear handling and serum sample collectionA total of 11 female black bears were captured and held in pens in the VirginiaTech Center for Bear Research from fall through spring, between the years 2006 and2009. Samples were collected as described in section 2.2.1.5.2.2 Cell cultureAll experiments were conducted using the osteoblastic cell line MC3T3-E1,subclone 4 (ATCC, Manassas, VA). This cell line displays markers of the osteoblastphenotype including high ALP, type I collagen, and OCN expression when cultured indifferentiation media (α-modified Eagle’s medium (α-MEM) with 10% fetal bovine serum(FBS), 50 μg/mL ascorbic acid and 10 mmol β-glycerophosphate) [268]. The cells werecultured in growth media (α-MEM with 10% FBS, and 1% penicillin/streptomycin) on100 mm culture dishes in a 5% CO 2 air atmosphere at 37°C. Media was replaced every48 hours, and cells were passaged when they reach 80% confluency. Cells were notused beyond the tenth passage.5.2.3 MC3T3-E1 apoptotic response to seasonal bear serumSeasonal MC3T3-E1 response to apoptotic threat was determined by a caspase-3 and -7 activity assay. Cells were seeded at 20,000 cells/well in tissue culture-treatedwhite-walled 96-well plates and allowed to attach overnight. Prior to starvation, cellswere primed for 24 hours in 2% FBS or bear serum. Since bear serum was collectedevery 10 days, several prehibernation, hibernation, and post-hibernation serum sampleswere available for each bear. Each well received a serum sample from a single bearfrom one collection day. Cells were starved in serum-free α-MEM for 6 hours, unlessotherwise noted. Media was aspirated and replaced with 50 μL PBS and 50 μLchemiluminescent DEVD substrate (Promega, Madison, WI), which releases photonsupon degradation by caspases 3 and 7. After 1 hour, luminescence was quantified by a72