Endothelial cell growth supplement from bovine neural tissue

HUVEC were isolated from human umbilical cords, obtained from the Royal London Hospital, using a modified method [25 (link),26 (link)]. In all experiments, HUVEC were used at passage 5, and the cell morphology was confirmed by phase contrast microscopy. HUVEC were cultured in HUVEC medium (Medium 199 supplemented with 150U/ml penicillin, 150U/ml streptomycin, 2mM L-glutamine (Sigma-Aldrich Ltd, Gillingham, Dorset, UK), 20% heat-inactivated FBS (GE Healthcare Europe GmbH, Vienna, Austria), 1U/ml heparin and 0.03mg/ml endothelial cell growth supplement from bovine neural tissue (Sigma-Aldrich Ltd). HEK 293T cells and the transformed human umbilical vein endothelial cell line, EaHy926, were maintained in DMEM (Sigma-Aldrich Ltd) supplemented with 10%FBS, 150U/ml penicillin and 150U/ml streptomycin. THP-1 cells were cultured in RPMI-1640 (Sigma-Aldrich Ltd) supplemented with 10%FBS, 50μM β-mercaptoethanol, 150U/ml penicillin and 150U/ml streptomycin.

Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (CRL-1730). HUVECs were cultivated in F-12K medium (ATCC) supplemented with 10% FBS (Gibco), endothelial cell growth supplement from bovine neural tissue (30 µg/mL) (Sigma–Aldrich, Poznań, Poland), and heparin (100 µg/mL) (Sigma–Aldrich, Poznań, Poland). HUVECs were seeded at a density of 6 × 10³ cells/cm² onto 24-well plates coated with rat tail collagen solution (Sigma–Aldrich, Poznań, Poland). Then 24-h cultures of HUVECs were exposed for 3 h to ACN and PP fractions at the concentrations of 0.1, 1, and 10 µg/mL and subsequently treated with TNF-α (10 ng/mL) (Sigma–Aldrich, Poznań, Poland) for an additional 3 h to induce inflammation.

Human umbilical vein endothelial cells (HUVECs) pooled from donors of both sexes were commercially obtained from Lonza or PromoCell. Cells were used within passage 5-6. Cells were maintained in HUVEC Growth Medium (HGM): M199 (Gibco, Life Technologies) supplemented with 20% Fetal Bovine Serum, (Labtech), 30 Μ g/m L endothelial cell growth supplement from bovine neural tissue (Sigma-Aldrich) and 10 U/m L Heparin (Sigma-Aldrich) and kept at 37°C. Mice stromal fibroblastic reticular cells (FRC) have been described previously (Acton et al., 2014 (link)) and were maintained in DMEM plus glucose (Life Technologies, Invitrogen) with 10% FBS, PS and 1% Insulin-Transferrin-Selenium (Life Technologies, Invitrogen).