Entactin collagen 4 laminin ecl cell attachment matrix

Human umbilical vein endothelial cells (HUVECs) (PromoCell GmbH, Heidelberg, Germany) were used to test the effect of the bacteria on vascular properties. After thawing, the frozen HUVECs were expanded in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO₂ in a humidifying incubator and used at passages p3 to p6 (30 (link), 44 (link)). Cells were grown to 80 to 90% confluence before being transferred to transparent polyethylene terephthalate (PET) Transwell supports (0.4 μm pore size, Greiner Bio-One, Austria), to a plastic well plate (Corning), to a glass-bottom well plate (Cellvis, Mountain View, CA), and to the insert chip (30 (link)). Before seeding, the uncoated substrates were treated with entactin-collagen IV-laminin (ECL) cell attachment matrix (Merck) diluted in Dulbecco’s modified Eagle’s medium (DMEM) (10 μg/cm²) for 1 h in the incubator. Then, the HUVECs, harvested with trypsin/EDTA solution (Biological Industries), were seeded inside the culture platforms at a density of 250,000 cells/cm² and grown for 48 h. Then, bacteria were added and their effect on cell behavior was tested after 1 h, 4 h, and 24 h.

Human umbilical vein endothelial cells (HUVECs; PromoCell GmbH, Heidelberg, Germany) were used to test the impact on cell-barrier properties. HUVECs were cultured in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO₂ in a humidifying incubator and used at passage p4–p6. Cells were cultured on transparent polyethylene terephthalate Transwell supports (0.4-μm pore size; Greiner Bio-One, Kremsmünster, Austria), coated with Entactin-Collagen IV-Laminin (E-C-L) Cell Attachment Matrix (Merck Millipore, Burlington, MA) diluted in Dulbecco's modified Eagle's medium (10 μg/cm²) for 1 h before seeding, at a density of 40,000 cells/cm², and grown for 3 days.

HUVECs (C-12200, PromoCell GmbH, Heidelberg, Germany, tested negative for mycoplasma contamination) were used to test each viral protein’s impact on vascular properties. After thawing, the HUVECs were expanded in low-serum endothelial cell growth medium (PromoCell) at 37°C with 5% CO₂ in a humidifying incubator, and used at passage p4–p6. Cells were grown to 80–90% confluence before being transferred to transparent polyethylene terephthalate Transwell supports (0.4 µm pore size, Greiner Bio-One, Austria) or a glass-bottom well plate (Cellvis, Mountain View, CA). Before seeding, the uncoated substrates were treated with Entactin-Collagen IV-Laminin (ECL) Cell Attachment Matrix (Merck) diluted in DMEM (10 µg/cm²) for 1 hr in the incubator. Then, the HUVECs were harvested using a DetachKit (PromoCell), were seeded inside the culture platforms at a density of 250,000 cells/cm², and grown for 3 days. Then viral infection with the different plasmids was performed and its impact on cell behavior was tested 3 days later.