Egm 2 medium

Human vascular endothelial cells (HUVECs), from PromoCell (C-12200) were mixed with dextran-coated Cytodex 3 microcarriers (GE) at a concentration of 400 HUVECs per bead in 1.5 ml of EGM-2 medium (Clonetics, Walkersville, MD). Beads with cells were shaken gently at 37 °C and 5% CO₂ every 20 min for 4 h. After incubating, beads with cells were transferred to a T2 tissue culture flask (Corning) and left overnight in 5 ml of EGM-2 at 37 °C and 5% CO₂. After that, beads with cells were washed three times with 1 ml of EGM-2 and re-suspended at a concentration of 200 cell-coated beads per ml with 2.5 mg ml⁻¹ of fibrinogen (Sigma) and 0.15 units ml⁻¹ of aprotinin (Sigma). Five hundred microlitre of fibrinogen/bead solution was added to 0.625 units of thrombin (Sigma) in one well of a 24-well tissue culture plate. The fibrinogen/bead solution was allowed to clot for 5 min at room temperature and then at 37 °C and 5% CO₂ for 20 min. One millilitre of EGM-2 (which contains 2% FBS) with or without 0.15 units ml⁻¹ aprotinin was added to each well and equilibrated with the fibrin clot for 30 min at 37 °C and 5% CO₂; 10⁶ macrophage cells were plated on top of the clot. Assays were terminated at day 7 for immunostaining and imaging55 (link).

Human pulmonary artery endothelial cells (HPAEC), primary cells isolated from the human pulmonary artery, were used for analysis. HPAEC were obtained from Clonetics BioWhittaker Inc. (Frederick, MD, USA). Cells were maintained in EGM-2 medium (Clonetics, BioWhittaker, Inc., Frederick, MD, USA) at 37 °C in atmosphere of 5% CO₂. We also used EA.hy926 vein endothelial cells. Originally, EA.hy926 cells were obtained by merging primary endotheliocytes isolated from human umbilical vein and cells of the thioguanine-resistant clone A549 (human lung carcinoma cells). Cells were grown at 37 °C and 5% CO₂ in DMEM (Dulbecco’s Modified Eagle Media) (PanEco, Moscow, Russia) with the addition of 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 2 mM glutamine (Hyclone, Logan, UT, USA) and 50 units/mL of penicillin-streptomycin (PanEco, Moscow, Russia). Experiments were performed in cultures at 6–10th passages. For modeling the endothelial barrier disruption, endothelial cells were stimulated with 0.01 µM nocodazole (Sigma, St. Louis, MO, USA). nocodazole stock solutions were prepared in DMSO. Final concentrations of DMSO in the cell medium did not exceed 0.1%.