10.1371/journal.pone.0008058.g004 Figure 4  Seeding of NB-like particles by fetuin-A, albumin, and serum in metastable versus supersaturated medium.
(A) Fetuin-A and albumin in both metastable and supersaturated media: bovine serum fetuin-A (BSF) and human serum albumin (HSA) were diluted into DMEM, separately or together, to the final concentrations indicated on the top. Where indicated, CaCl2 and NaH2PO4 were added to the final concentrations shown at the bottom (labeled as “Calcium+Phosphate Added”), followed by incubation in cell culture conditions for “1 Week” or “1 Month”, as indicated on the right of each panel. “None” refers to the absence of any exogenously added calcium and phosphate, that is, to metastable medium DMEM. Seeding of the proteins alone without additional ion input did not produce any turbidity change even following incubation for 1 month (“None” column). Low amounts of precipitation were noticed when precipitating ions were added (supersaturated solutions) that increased gradually over time through either bell-shaped (BSF, BSF and HSA) or straight, dose-dependent (HSA) relationships. (B) Serum in supersaturated medium: FBS (top panel) or HS (lower panel) were added to the final concentrations indicated on the top, followed by addition of precipitating reagents to the concentrations shown at the bottom. Addition of precipitating reagents at either 0.1, 0.3, or 0.5 mM into DMEM containing FBS or HS produced turbidities which increased steadily as seen after “1 Week” either through bell-shaped (0.1 mM calcium and phosphate) or straight, dose-dependent (0.3 and 0.5 mM calcium and phosphate) relationships. After further incubation, all treatments showed straight dose-dependent turbidities that continued to increase in a slow and gradual manner (“1 Month”).