Quantification of Extracellular Calcium Deposition

To quantify the amount of extracellular calcium deposition, the cells were subjected to Alizarin Red S staining as published previously [26 (link),27 (link)]. In short, cells were fixed in 70% ethanol (Carl Roth, Karlsruhe, Germany), incubated over night at 4 °C, washed with Aqua dest. and then stained with 0.5% Alizarin Red S solution (Waldeck, Münster, Germany) for 10 min. After washing with PBS, a 10% hexadecylpyridinium chloride solution (Merck, Darmstadt, Germany) was added to each sample and incubated on an oscillator (IKA-Werke, Staufen, Germany) for 30 min at 350 rpm to dissolve the stained calcium depositions. After complete dissolution each sample was measured spectrometrically at 570 nm as technical duplicates and normalized to a standard curve.