Primary mouse aortic smooth muscle cells (MAoSMCs) were isolated from Asm−/− and Asm+/+ mice and routinely cultured (passages from 3 to 7) as described previously [50 (link)]. MAoSMCs were treated with 2 mM β-glycerophosphate (Sigma–Aldrich) and/or 0.01 U/ml sphingomyelinase (Staphylococcus aureus) (Enzo Life Sciences). For analysis of mineralization, treatment with calcification medium containing 10 mM β-glycerophosphate and 1.5 mM CaCl2 (Sigma–Aldrich) was used. For long-term experiments, fresh medium with agents was added every 2–3 days.
Protocol detail
Aortic Smooth Muscle Cell Mineralization
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Aortic
Calcification
Mice
Mineralization
Smooth muscle cells
Sphingomyelinase
Staphylococcus aureus
β glycerophosphate
Corresponding Organization : German Centre for Cardiovascular Research
Other organizations : University of Tübingen, Innsbruck Medical University, Universität Innsbruck, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Deutsches Herzzentrum der Charité, University of Duisburg-Essen
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Variable analysis
independent variables
- Genotype (Asm-/- and Asm+/+ mice)
- Treatment with 2 mM β-glycerophosphate
- Treatment with 0.01 U/ml sphingomyelinase (Staphylococcus aureus)
- Treatment with calcification medium containing 10 mM β-glycerophosphate and 1.5 mM CaCl2
- Mineralization
- Passage number of primary mouse aortic smooth muscle cells (MAoSMCs) (passages from 3 to 7)
- Culture conditions as described previously [50]
- Positive control: Treatment with calcification medium containing 10 mM β-glycerophosphate and 1.5 mM CaCl2
- Negative control: Unstated, but may be untreated MAoSMCs or MAoSMCs from Asm+/+ mice
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