Eclipse ti e inverted microscope

HUVECS were cultured, as described above, in 35-mm 4-well CELLview glass-bottom dishes (Greiner Bio-One 627870) coated with gelatin. Cells were infected with LifeAct-GFP lentivirus for 2 d and then transduced again with either VE-cadWT-RFP or VE-cadDEE-RFP adenovirus. 8 h after adenoviral infection, cells were starved overnight in EBM-2 basal medium containing 1% FBS. Cells were manually scratched with a p200 pipette tip the next morning, and the starvation medium was replaced with phenol red–free growth medium. Dishes were placed on the microscope stage and maintained at 37°C in 5% CO₂ using a humidified temperature/CO₂-controlled chamber (Tokai Hit). Cells were imaged using a Nikon Eclipse Ti-E inverted microscope (60x/1.49-NA Apo TIRF oil-immersion objective) equipped with a motorized stage and a Hamamatsu C11440-22CU digital camera. Images were taken once per minute for 2 h, beginning 1 h after cells were scratched. Endocytic budding from the rear of wound-edge cells was quantitated by a blinded observer. At least eight videos were analyzed per condition, and significance was evaluated using a t test.

Primary mouse endothelial cells were plated in growth medium in 35-mm Culture-Insert 3-well wounding assay dishes (Ibidi 80366) and grown to confluence. Cells were then starved overnight in EBM-2 basal medium (Lonza cc-3156) containing 1% FBS. The next morning, the silicone insert was removed, leaving a 500-µm wound. Each field was imaged by phase contrast with a 10× dry objective (0.3 NA) at initiation of wound closure and the indicated time points to monitor wound closure. Images were obtained using a Nikon Eclipse Ti-E Inverted Microscope equipped with a motorized stage and a Hamamatsu C11440-22CU Digital Camera and NIS-Elements software version AR4.40.00. Cells were maintained at 37°C in 5% CO₂ during imaging. Wound area was measured using ImageJ. A D’Agostino–Pearson normality test was performed to confirm normal distribution of the data, and a two-tailed t test was used to evaluate significance at each time point.