Recombinant human or mouse tnf α

The human umbilical vein endothelial cell line EA.hy926 cells was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and primary mouse aortic endothelial cells (MAECs) were purchased from Lifeline Cell Technology (LifeScan, Walkersville, USA). Both EA.hy926 cells and MAECs were stimulated with recombinant human or mouse TNF-α (100 ng/mL, R&D Systems, Minneapolis, USA) for 24 hours to produce EMPs. The cell supernatants were collected and centrifuged at 1000 × g for 10 min and then at 3000 × g for 5 min to remove cells and large debris. The cell-free supernatant was centrifuged at 16000 × g for 60 min at 4°C to pellet EMPs. The obtained EMPs were washed in sterile phosphate-buffered saline (pH 7.4) and pelleted again at 16,000 × g for 60 min. Quantification of the EMPs was performed using the Bradford method [24 (link)].