Smooth Muscle Cell Culture and Characterization

Human ASM cells were obtained from ScienCell Research laboratories. Cells were cultured in six-well plates in Smooth Muscle Cell Medium (SMCM) containing 10% FBS and were maintained at 37 °C and 5% CO₂ as previously described [13 (link)]. Cells from passage 3–6 maintained their SMC phenotype and were used in all experiments. ASM cells were characterized by smooth muscle cell markers including smooth muscle α-actin and smooth muscle heavy chain using immunofluorescence. In inhibition experiments, inhibitors of signal transduction pathways were added for 2 h before the addition of VEGF. All inhibitors were dissolved in dimethyl sulfoxide (DMSO; final concentration of 0.1%, vol/vol) and added to the medium. Vehicle controls contained the same amount of DMSO.