Egm 2 kit cc 4176

HUVECs were seeded at a density of 6.0 × 10⁴ cells/well on the luminal side of filters (0.4 μm pore size; Corning) coated with 1% gelatin in 12-well plates. Cells were grown in EC basal medium (EBM-2, CC-3156) containing EGM-2-kit (CC-4176) (Lonza Walkersville, Inc., MA, United States) and 10% fetal bovine serum at 37°C in humidified 5% (v/v) CO₂. Cells were cultured for 2 days until confluent, and starved in serum-free medium for 2 h and treated with PD (5 µM) for 30 min before induction with VEGF (30 ng/ml; Komabiotech) for 30 min. Transendothelial electrical resistance (TEER) was measured using a chop-stick electrode (World Precision Instruments STX2) with Millicell ERS-2 volt/Ω m (Millipore, MA, United States). The TEER of the cell-free gelatin-coated filters was subtracted from the measured TEER and are given as Ω × cm². Paracellular vascular permeability was also confirmed using fluorescein isothiocyanate (FITC)- dextran fluorescein. FITC-dextran (30 mg/ml; Sigma) was added to the upper compartment. The absorbance of the lower chamber solution was measured at 492 nm (excitation) and 520 nm (emission) using a FLUOstar Omega microplate reader.

Human umbilical vein endothelial cells were seeded at a density of 4 × 10^5 cells/well onto 12-well Transwell semipermeable supports (0.4 μm pore size; Corning) coated with 1% gelatin. HUVEC were cultured in EC basal medium (EBM-2, CC-3156) containing EGM-2-kit (CC-4176) (Lonza Walkersville, Inc., MA, United States) and 10% FBS at 37°C in a 5% CO₂ incubator in a humidified atmosphere. Upon confluence, the cells were starved in serum-depleted medium for 2 h and treated with 100 kilounits/mL IL-2 for 4 h. Endothelial cell permeability was confirmed using fluorescein isothiocyanate (FITC)-dextran fluorescein. FITC-dextran (30 mg/mL; Sigma-Aldrich) was added to the upper chamber and incubated for 30 min. The absorbance was measured at 492 nm (excitation) and 520 nm (emission) using a FLUOstar Omega microplate reader. The transendothelial electrical resistance (TEER) assay was performed using a chopstick electrode (World Precision Instruments STX2) with Millicell ERS-2 volt/Ω m (Millipore, MA, United States) and the results expressed as Ω × cm².

Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Basel, Switzerland). Cells were grown in 2% gelatin-coated dishes and maintained in medium 199 (Invitrogen, CA, United States) containing 20% fetal bovine serum (HyClone, Tianjin), 1% penicillin/streptomycin, 3 ng/ml basic fibroblast growth factor (R&D system, Minneapolis), and 5 U/mL heparin (Sigma-Aldrich, MO, United States) at 37°C in humidified 5% (v/v) CO₂ atmosphere. Human retinal endothelial cells (HRECs) were purchased from Cell Systems Inc. (Kirkland, WA, United States). Cells were grown in 2% gelatin-coated dishes and maintained in EC basal medium (EBM-2, CC-3156) containing EGM-2-kit (CC-4176) (Lonza Walkersville, Inc., MA, United States) and 20% fetal bovine serum at 37°C in humidified 5% (v/v) CO₂. Cell passages between 3 and 6 were used for experiments.