Cytotoxic effects of CaP particles in the presence or absence of fetuin-A or albumin or functionalised CaP particles (CaP with fetuin-A attached, CaP/F or with albumin attached, CaP/A) on fura-2 loaded VSMCs.

Stock concentrations of particles contained 2.9 mg/mL Ca2+ for CaP, 0.8 mg/mL Ca2+ for CaP/F and 1.84 mg/mL Ca2+ for CaP/A. The particle concentration used in each experiment was 25 µg/mL in terms of Ca2+ content. Samples were prepared as a 250 µg/mL solution in 100 µl physiological buffer and added to VSMCs in a chamber containing 900 µl physiological buffer. All particle solutions were vortexed immediately prior to addition to cells. Raw data are presented, i.e. 82/102 denotes that 82 out of 102 cells that were imaged died within 1 hour of an experiment. Cell death was determined by fura-2 leak from cells. ‘n’ represents the number of separate experiments. Representative Ca2+ traces are shown in figure 2 and figures S2, S4 and S5.