Measuring expression of adhesion molecules

ECs, 2·5 × 10⁴, were grown to 90% confluence in six‐well plates at 37°C/5% CO₂; 10 ng/ml of TNF‐α was added for 24 h. Cells were removed from the plate using Accutase before being incubated with Fc block (anti‐CD16/32 antibody; Affymetrix eBioscience, Hatfield, UK) for 30 min at 4°C. Cells were then stained with anti‐ICAM‐1 or anti‐vascular cell adhesion protein 1 (VCAM‐1) fluorescein isothiocyanate (FITC) antibodies or isotype controls (Affymetrix eBioscience) before being analysed by flow cytometry and FlowJo software.