Primovert inverted microscope

Prior to seeding, 96-well plates were coated with 50 μL/well of Matrigel (Corning) and incubated at 37 °C for 30 min. PAECs were then seeded at 20,000 cells/well in the growth medium and further incubated at 37 °C for 6–8 h. Images were taken for each well every 1 h after 4 h using a Zeiss Primovert Inverted Microscope (Zeiss) attached to a Zeiss Axiocam ERc 5 s (Zeiss), to determine the optimal time point for the quantitative analysis. Images in low magnification (×4) were captured for each well. Each image was divided into 16 rectangles, and 10 rectangles were randomly chosen. The lengths of tubes between the branching junctions were measured and summed in each single rectangle, and then values obtained from 10 rectangles were averaged. A measurement example is shown in Supplementary Fig. 9. We performed experiments 3 independent times using 1–2 wells for each group.

For endothelial cell migration analysis (44 (link), 45 (link)), EA.hy926 cells were plated in 24-well plates at a density of 8 × 10⁴ cells/well. When cells reached 90% confluency, they were synchronized in DMEM/antibiotics medium for 16 h and then cell monolayers were scratched using a pipette tip (200 μl). Wounded cells were rinsed with warm DPBS to remove nonadherent cells and then EA.hy926 cells were incubated with 0.35 μM (at apoA-I concentration) human HDL (apoB-depleted plasma), for 1 h at 37°C with or without 2.5 μg/ml of blocking antibody for PCSK9, in DMEM/antibiotics medium for 24 h. At the end of the incubation period, cells were fixed with ice-cold methanol for 15 min, permeabilized in 0.1% Triton X-100/DPBS for 15 min, and stained with hematoxylin (Sigma Aldrich) for 5 min. Cells were photographed on marked positions at 0 and 24 h, using the 10x objective lens of a Zeiss Primovert Inverted microscope (Zeiss, Germany). Wound healing was quantified, following analysis of images with ImageJ analysis software (NIH, Bethesda, MD) (46 ), by measuring the number of cells that migrated.