Figure 3
Determination of calcification in the MOVAS-1 cell line at various concentrations (3.0 mM Pi, 5.0 mM Pi, 2.4 mM Ca and 1.4 mM Pi, 2.7 mM Ca and 2.5 mM Pi). Monolayers of MOVAS-1 cells were grown to confluence (day 0) and then switched to calcification media. The monolayers were incubated in calcification media (3.0 mM Pi, 5.0 mM Pi, 2.4 mM Ca and 1.4 mM Pi, 2.7 mM Ca and 2.5 mM Pi) for 7 days, changing media on alternate days. (A) Calcium leaching assay: o-cresolphthalein complexone. (B) Fluorescence assay: cell monolayers were incubated with Fluorescein-BP (1 μM, 2 hours); NBF (10%, 15 minutes). (C) Fluorescence images and Alizarin S stain images: cell monolayers were either incubated with Fluorescein-BP (1 µM, 2 hours) and then fixed with NBF (10%, 15 minutes), or fixed with NBF (10%, 15 minutes) and then incubated with Alizarin S (2%, 500 μL, 10 minutes). Data shown in (A) and (B) are from at least 6 repeats and shown as the mean ± S.E.M., *P < 0.05, **P < 0.01, ***P < 0.001 compared to control, n = 6. Images shown in (C) are representative of at least 3 independent experiments yielding comparable results. Arrows indicate the location of calcium deposits within the well. Scale bars = 250 μm.
