Quantifying Vascular Smooth Muscle Cell Calcification

HAoSMCs were incubated overnight at 37 °C with OsteoSense 680EX (1:250, Perkin Elmer, Traiskirchen, Austria) [42 (link)]. Images were acquired with the ChemiDoc MP imaging system (Bio-Rad Laboratories, Vienna, Austria) with excitation/emission (bandpass) wavelengths of 680/715(30) nm [43 (link)]. HAoSMCs were decalcified in 0.6 M HCl at 4 °C overnight and the calcium content was quantified with the QuantiChrom Calcium assay kit (BioAssay Systems, Hayward, CA, USA) [14 (link),21 (link),37 (link)]. Proteins were isolated using 0.1 M NaOH/0.1% SDS buffer and quantified by the Bradford assay (Bio-Rad Laboratories, Vienna, Austria). Data are shown normalized to total protein concentration.