A488 anti higg

ARPE-19 cells (ATCC) were incubated for 30 min with serial dilutions of soluble heparin (Sigma-Aldrich; starting from 1000 nM, 1:2) or rhNRP-1-mFc (Regeneron; starting from 100 nM, 1:2) pre-incubated with 10 nM VEGF₁₆₅ (Regeneron). Bevacizumab was added to the cells (final concentration 10 nM), followed by a 1-h incubation at 37 °C. Surface staining of bevacizumab was detected with A488-anti-hIgG (Jackson Immunoresearch), followed by fixation in 4 % paraformaldehyde and counterstaining with DAPI. Fluorescence was detected by a Molecular Devices ImageXpress Micro XL High-Content Imaging System equipped with a Nikon 20x Plan Fluor ELWD objective lens (NA 0.45; WD 8.2–6.9 mm). All images were acquired with a Molecular Devices 1.4 megapixel cooled CCD camera using MetaXepress^® High-Content Image Acquisition and Analysis Software and analyzed using PerkinElmer Columbus Image Data Storage and Analysis System. Figures were made in Adobe Photoshop. No adjustments were made to the original images.

Serial dilutions of heparin (Sigma-Aldrich) or rhNRP-1-mFc (Regeneron) were premixed with 10 nM VEGF₁₆₅. The mixture was then added to HUVEC plated as described above and incubated for 30 min at 37 °C. A constant concentration of 15 nM bevacizumab was then added to cells, followed by a 1-h incubation at 37 °C. The cells were washed, detected with 1 μg/mL A488anti-hIgG (Jackson), counterstained with Draq5 (Cell Signaling Technologies), and imaged using Molecular Devices ImagExpress Micro XL High-Content Imaging System equipped with a Nikon 20x S Plan Fluor EL WD objective lens (NA 0.45; WD 8.2–6.9 mm). All images were acquired with a Molecular Devices 1.4 megapixel cooled CCD camera using MetaXepress^® High-Content Image Acquisition and Analysis Software and analyzed using PerkinElmer Columbus Image Data Storage and Analysis System. Figures were made in Adobe Photoshop. No adjustments were made to the original images.