Clear bottom 96 well tissue culture plates

HMVEC-D were seeded on BD-Falcon clear-bottom 96-well tissue culture plates (catalogue number 353219) at a density of 10,000 cells per well. Cells were incubated with denoted treatments for 5 hr and then fixed with 4% paraformaldehyde in PBS for 10 min. Cells were then permeabilized with 0.5% triton-X 100 for 2 min, blocked with Odyssey™ Blocking Buffer (Licor Biosciences) for 30 min at room temperature, and incubated with a mouse monoclonal VE-Cadherin antibody (BD, 555661) over night at 4°C. Cells were then rinsed with PBS and incubated with Alexa Fluor® 488 donkey anti-mouse secondary and Hoechst 33258 dye for 4 hr at room temperature. Cells were rinsed and the Molecular Devices ImageXpress Micro XLS collected nine non-overlapping images at the center of each well with a 20X objective. VE-cadherin area of each image was quantified with ImageJ by batch thresholding all images from an individual experiment and quantifying the number of pixels that were VE-Cadherin positive within each image. VE-cadherin area was then divided by the number of nuclei per image and then normalized to the mock-treatments. Data are compiled from three independent experiments with three replicate wells per condition in each experiment and composed of nine images per well for a total of 81 images per condition.