Figure 6. Seeding of NB-like particles by fetuin-A and albumin at low concentrations in supersaturated ionic solutions.

Stock solutions of BSF and/or HSA were diluted into DMEM to the relatively low concentrations shown on the top. The precipitating reagents CaCl₂ and NaH₂PO₄ were then added at either 0.3, 0.5, or 0.7 mM, as indicated at the bottom (labeled as “Calcium+Phosphate Added”). Turbidity was monitored by A₆₅₀ turbidity reading following inoculation (“Day 1”) and after incubation in cell culture conditions for the time indicated on the right side of each panel. Bell-shaped curves of precipitation were noticed at “Day 4” for the three different combinations of proteins as well as the three different concentrations of added precipitating reagents. Precipitation in these conditions increased in a time-dependent manner with further incubation. When 0.3 mM of precipitating reagents was used, bell-shaped increases in turbidity were seen for the three protein combinations after 1 month of incubation. With calcium and phosphate added to 0.5 and 0.7 mM, the initial bell-shaped increase in turbidity observed at “Day 4” was seen to shift to the right at “1 Month” reading.