2.6. Collagen synthesis

Passage 1 hAF cells were seeded at a seeding density of 5000 cells/well in 24‐well cell culture plates in Ham's F‐12 medium supplemented with 10% FBS and 1% P/S (0.5 mL media/well). Cells were grown to 70% to 80% confluency and then treated with polyP‐22 supplementation in varying concentrations (0, 0.5, and 1.0 mM). At the same time, 10 μCi/mL ³H‐L‐proline in 0.5 mL F‐12 was added for 48 hour. The medium was collected, and the cell samples were scraped in in homogenization buffer (0.5 mL/well) and collected and subjected to three freeze/thaw cycles. The samples were stirred at 4°C overnight, combining the medium and cell layers, then collagen synthesis was determined by ³H‐L‐proline incorporation using a modified collagenase digestion method as previously described. ³⁵ One milliliter of the supernatant and aliquots of the pellets dissolved in 0.2 mol/L NaOH were subjected to scintillation counting to determine the collagen and noncollagen protein synthesis. ³⁶ Raw counts per minute were normalized to DNA content (ng) determined using the Quant‐iT PicoGreen ds DNA Assay Kit (Invitrogen).