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Supplemental Material Demyanets et al. � Supplemental Methods � Supplemental Tables I, II, III � Supplemental Figures I, II, III Supplemental Methods Isolation <strong>of</strong> human polymorph-nuclear leukocytes (PMNL) Human polymorph-nuclear leukocytes (PMNL; 99% neutrophils, less than 1% eosinophils) were isolated from heparinized (100 U/mL) peripheral venous blood <strong>of</strong> healthy donors as described. 1 Red blood cells were sedimented with 6% dextran (Pharmacia, Uppsala, Sweden) in Hank’s balanced salt solution (HBSS; Sigma, St. Louis, MO, USA) for 60 min at room temperature, leukocyte-rich plasma was collected <strong>and</strong> PMNL were then isolated by centrifugation, washed <strong>and</strong> resuspended to 1x10 6 cells/mL in RPMI-1640 (Sigma) with 10 mmol/L HEPES (Sigma). <strong>Adhesion</strong> assay under static conditions PMNL adhesion to HCAEC or HUVEC was measured as described. 1 Briefly, endothelial cells were plated in gelatine coated 24-well plates <strong>and</strong> allowed to grow to confluence. The medium was removed <strong>and</strong> cells were then incubated with 1.0 mL/well <strong>of</strong> minimum essential medium (M199; Sigma) containing 20% fetal calf serum (FCS, Lonza, Verviers, Belgium) with or without recombinant human (rh) IL-<strong>33</strong> (R&D Systems; Minneapolis, MN). After 4 h <strong>of</strong> incubation the culture supernatant <strong>of</strong> such treated cells was removed <strong>and</strong> the cells were gently washed three times with M199. 1.0 mL <strong>of</strong> HBSS containing 1 x 10 6 PMNL was then added to the endothelial cell monolayer in each well. The binding phase <strong>of</strong> the assay was performed at Downloaded from http://atvb.ahajournals.org/ at Bibliothek der MedUniWien (IX0000096057) on September 2, 2011
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