2.6. Smooth muscle cell isolation and proliferation

Smooth muscle cell isolation and proliferation assays were performed as described [26]. SMC were obtained by outgrowth from explants of WT and Nf1^+/− thoracic aortas. SMC were cultured in DMEM supplemented with 20% fetal bovine serum and 100 U/ml penicillin/streptomycin in a 37 °C, 5% CO₂-humidified incubator. For cell proliferation, SMC (5000 cells/cm²) were placed in a 96-well plate and deprived of growth factors for 12–18 h. Quiescent SMC were stimulated with H₂O₂ (1 and 100 μM) for 24 h and pulse-labeled with 1 μCi/ml of [³H] thymidine for 6 h. β emission was measured and reported as counts per minute. Cell counts using a hemocytometer were performed to confirm radioisotope results. Under the same conditions, cell viability was assessed by MTT assay and light absorbance was measured using a plate reader (570 nm). All experiments were performed in triplicate in four distinct cohorts.