Complete mouse endothelial cell medium w kit

Primary CEC were isolated from adult 3–4 and 18–20 m/o old C57BL/6J mice using the Adult Brain Dissociation Kit according to the manufacturer’s instructions (MACS Miltenyi Biotec, #130-107–677). Briefly, mouse brains were homogenized and enzymatically digested. Homogenized brains were filtered through 70 µm-pore strainers, and debris was separated by centrifugation (3,000 × g, 10 m). Cellular suspensions were then treated to remove red blood cells. Finally, cellular suspension was plated in pre-coated flasks with 0.1% sterile porcine gelatin (Biological Industries, #01-944-1B). Cells were maintained in Complete Mouse Endothelial Cell Medium/w Kit (Cell Biologics, #M1168) supplemented with puromycin (4 μg/ml) for 48 h to select endothelial cells (Perriere et al., 2005 (link)). After 48 h, cells were washed with PBS to remove puromycin and debris. Cells were then maintained in fresh Complete Mouse Endothelial Cell Medium until 100% confluence.

Both the murine retinal photoreceptor cell line 661W and Vero cells were obtained from Osaka Bioscience Institute. Both 661W and Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 with l-glutamine and phenol red (661W) (Fujifilm Wako Pure Chemical Corporation) or high-glucose DMEM with l-glutamine, phenol red, HEPES, and sodium pyruvate (Vero) (Fujifilm Wako Pure Chemical Corporation), supplemented with 10% heat-inactivated foetal bovine serum (FBS) (Thermo Fisher Scientific) in a humidified incubator with 5% CO₂. Retinal microvascular endothelial cells of BALB/c mice were obtained from Cell Biologics and were cultured in complete mouse endothelial cell medium/w Kit (Cell Biologics) supplemented with 10% heat inactivated FBS (Thermo Fisher Scientific) in a humidified incubator with 5% CO₂.