Split kit

Human aortic endothelial cells (HAoEC) were purchased from PromoCell, Germany. HAoEC were cultured strictly following the manufacturer's recommendations using Endothelial Cell Growth Media MV2 and the PromoCell Split Kit. HAoEC were used up to passage 9. The media was supplemented with the supplement mix provided by the manufacturer, which contains 4% FBS and 2.5 μg/ml free cholesterol.

Primary human aortic endothelial cells (HAoEC, PromoCell, Germany) were cultured according to manufacturer’s recommendation using the corresponding media as well as the split kit (all, PromoCell, Germany). Cells were used up to passage 8. HAoECs were stimulated for 2 hours with 50 ng/ml IFN-γ, 10 ng/ml TNF-α, or 50 ng/ml IFN-γ + 10 ng/ml TNF-α. Subsequent to the 2-hour incubation, the HAoECs were harvested and RNA was extracted using a Directo-zol RNA MiniPrep (Zymo Research). The extracted RNA was converted to cDNA utilizing the Qiagen RT² Strand Kit and gene expression was analyzed on custom RT-PCR plates for endothelial cell activation markers (Qiagen). Gene expression was reported as fold-change relative to untreated HAoECs.