Primary SMCs were isolated from aneurysmal and control tissue of the ascending aorta as described in more detail elsewhere (11 (link), 19 (link)). Cells were cultivated in specific SMC medium (Medium 231 supplemented with SMC Growth Supplement; Gibco, Thermo Fisher Scientific, Austria) at 37°C until reaching passage 3 before being used for experiments. All SMC samples were tested for contamination with endothelial cells and fibroblasts by Western blot analyses using an anti-CD90 antibody for fibroblasts (Dianova, Germany, #DIA-100) and an anti-CD31 antibody for endothelial cells (Dako, United States, #M082301).
For metabolomic analyses, trypsinized cells were counted and 1x106 cells per sample were added to 100 μl ice cold ultrapure absolute Ethanol (Carl Roth, Karlsruhe, Germany) for cell lysis. Samples were sonicated (15 intervals, cycles: 10%, power: MS72/; Sonopuls HD 200; Bandelin, Berlin, Germany) and all solid components were separated by centrifugation at 10,800 g at 4°C for 10 min. Supernatants were collected and stored at −80°C for further analyses.
For metabolomic analyses, trypsinized cells were counted and 1x106 cells per sample were added to 100 μl ice cold ultrapure absolute Ethanol (Carl Roth, Karlsruhe, Germany) for cell lysis. Samples were sonicated (15 intervals, cycles: 10%, power: MS72/; Sonopuls HD 200; Bandelin, Berlin, Germany) and all solid components were separated by centrifugation at 10,800 g at 4°C for 10 min. Supernatants were collected and stored at −80°C for further analyses.
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