Hcmec

As detailed previously 2, 13, blood was obtained from healthy volunteers under a protocol approved by the Institutional Review Board of Baylor College of Medicine. ACD (Sigma) anticoagulated blood was fractionated by Ficol‐Hypaque gradient centrifugation (Histopaque‐1077; Sigma) to collect mononuclear cells (MNL). For a TEM assay, human cardiac microvascular endothelial cells (HCMEC) (Lonza (Walkersville, MD), passages 4–9) were seeded onto an insert coated with human collagen type IV (Millipore, Temecula, CA). Human MNL (in RPMI1640 with 10% FBS and antibiotic–antimycotic (Invitrogen) or without serum but with ITS‐X (Sigma–Aldrich)) were then added to each insert and the same medium including 650 ng/mL MCP‐1 for chemoattraction was added to the well below and the indicated stimuli to the insert. MNL were allowed to migrate for 16 or 96 h before fixing and counting the cells on the bottom of the well. At the end of each migration period, the migrated cells were harvested immediately for RNA or, for protein, after 6 h of exposure to Brefeldin A (Sigma).