Ecgs heparin

Human umbilical vein endothelial cells (HUVECtert) were cultured in Nunclon^TM Delta surface flasks (Bartelt; Graz, Austria) in medium 199 (Gibco; Carlsbad, CA, USA) supplemented with 20% fetal calf serum (FCS) (Sigma-Aldrich; St. Louis, MO, USA), penicillin-streptomycin-amphotericin B (Lonza; Basel, Switzerland), and ECGS-heparin (PromoCell; Heidelberg, Germany) in the 95% humidified atmosphere with 5% CO₂ at 37 °C. Stimulation of cells was performed in endothelial basal medium-2 (EBM-2) (Lonza) supplemented with 2% FCS (Sigma-Aldrich), and 20 mM HEPES (Lonza). The cells were preincubated with an inhibitor solution for 20 min. Then, an agonist solution was added to the cells. After 7 h of the incubation at 37 °C, cell culture supernatants were collected for ELISA. At the end of the experiment, viability of the remaining cells was measured using a tetrazolium salt (XTT) assay. Following inhibitors and agonists used were purchased from Sigma-Aldrich: AZ11645373, A740003, Ac-YVAD-cmk, LPS 005:B5, ATP, BzATP. Recombinant human TNFα was from PeproTech (Rocky Hill, NJ, USA).

Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords using collagenase treatment essentially as described (Jaffe et al., 1973 (link); Zhang et al., 1997 (link)). The studies were reviewed and approved by Ethics Commission of the Medical University of Vienna. Written informed consent was provided by the participants’ legal next of kin. Cells were seeded in 75 cm² flasks coated with 1% gelatin (Sigma, St. Louis, MO, United States, #04055) and cultured in M199 medium (Lonza, Basel, Switzerland, #12-119F) with 20% heat-inactivated FBS (Sigma, St. Louis, MO, United States, #F6765), penicillin (100 units/ml), streptomycin (100 μg/ml), (Pen-Strep, Lonza, Basel, Switzerland, #DE17-602E), 2 mM L-glutamine (Sigma; #G7513), 5 units/ml heparin, and 25 μg/ml ECGS (Promocell, Heidelberg, Germany, ECGS/Heparin #C-30140). Cells were passaged at a ratio of 1:3 and used until passage 5 for experiments.

Human endothelial cells (HUVEC) were isolated from the veins of umbilical cords as described previously [19 (link)], and maintained in M199 medium (Lonza, Basel, Switzerland, #12-119F) supplemented with 20% FCS (Sigma, St. Louis, MO, USA, #F6765), 2 mM L-glutamine (Sigma; #G7513), penicillin (100 units/mL), streptomycin (100 μg/mL), (Pen-Strep, Lonza, Basel, Switzerland, #DE17-602E), 5 units/mL heparin, and 25 μg/mL ECGS (Promocell, Heidelberg, Germany, ECGS/Heparin #C-30140). For experiments, post-confluent cells of passage no greater than 5 were used.