Isolation and Senescence of Mouse Aortic VSMCs

As described before,19 (link) primary VSMCs from the mouse aorta were extracted. To obtain primary VSMCs, thoracoabdominal aortas were extracted from healthy C57BL6 mice. The collected tissues were washed with DMEM (BasalMedia, Shanghai, China) and cut into 1 mm³ pieces. The pieces were plated onto 24-well plates and digested with 1 mg/mL collagenase (Sigma-Aldrich) and 0.5 mg/mL elastase (Sigma-Aldrich) for 1.5 h at 37°C. Collected VSMCs were cultured in DMEM with 100 U/mL penicillin, streptomycin, and 10% FBS (BasalMedia) in a humidified atmosphere containing 5% CO₂. Cells from passages to 3–5 were used for subsequent analyses. VSMCs were treated with 50 μg/mL oxidized low-density lipoprotein (ox-LDL; Solarbio, China) for 72 h of incubation at 37°C to induce VSMC senescence.