Figure 3. Timing of fetuin-A addition affects the response to CaP particles.

Pre-incubating with fetuin-A (1 µM) for 15 minutes followed by replacing cell supernatant with fetuin-A-free physiological buffer gave no protection against cell death on CaP exposure (12.5 µg/mL) (A and B). The ratio of 340/380 fura-2 values is plotted in A and raw data is presented in B, showing that when 340 and 380 nm values both suddenly decline together (fura-2 leak from VSMC), this coincides with PI influx. The Ca²⁺ activity observed up to the time of fura-2 loss/PI influx represents genuine Ca²⁺ signals, whereas activity seen after this time point (in A) is not genuine. (C) Simultaneous addition of CaP (12.5 µg/mL) and fetuin-A (1 µM) had very little effect on VSMC intracellular Ca²⁺ and cells survived over one hour of analysis. Representative traces are shown.