Microvascular endothelial cell growth kit vegf

Human telomerase (hTERT)-immortalized microvascular endothelial cells isolated from human foreskin (TIME cells; ATCC) were grown in ATCC’s vascular cell basal medium supplemented with microvascular endothelial cell growth kit-VEGF and 12.5 μg/ml blasticidine (Sigma-Aldrich) for 48 h at 37°C + 5% CO₂ until reaching 80–90% confluence. At confluence, cells were passaged and 5.4 × 10⁴ cells were plated on fibronectin-coated (10 µg/ml; MilliporeSigma), base/acid cleaned, 0.17 mm (#1.5) glass-bottom dishes (14 mm glass diameter; MatTek) for 18 h prior to experiments.

Human Telomerase (hTERT)-immortalized microvascular endothelial cells isolated from human foreskin (TIME cells, ATCC, Manassas, VA), or TIME cells stably expressing mNeonGreen-Actin (TIME-mNGrActin; see description of cell line generation below), both at passage 16 or higher (unless indicated otherwise), were grown in ATCC’s vascular cell basal medium supplemented with microvascular endothelial cell growth kit-VEGF and 12.5 μg/mL blasticidine (Sigma-Aldrich, St. Louis, MO, 203351) for 48 h at 37°C + 5% CO₂ until reaching 80–90% confluency. Then cells were passaged and plated on fibronectin (FN)-coated (10 μg/ml, MilliporeSigma, Burlington, MA), base/acid cleaned, 0.17 mm (#1.5) glass bottom dishes (14 mm glass diameter, MatTek, Ashland, MA) or 6 well plates for 18 h prior to experiments.