Vasculife complete medium

HUVECs are widely used to study endothelial injury in vitro [15 (link)]. HUVECs (Lifeline Cell Technology, Walkersville, MD, USA) were cultured in VascuLife complete medium (Lifeline Cell Technology) at 37 °C in a humidified 5% CO₂ incubator. The cells at passages 4–10 were used in these experiments. For hypoxic exposure, the cells were maintained in VascuLife basal medium supplemented with 2% fetal bovine serum and exposed to hypoxic conditions (1% O₂) in a humidified 5% CO₂ hypoxic chamber (Astec, Fukuoka, Japan).

Endothelial cell lines were obtained from lungs of WT, CSA KO and CX mice strains to assess EC proliferation capacity, migration and ICAM1 activation in vitro. For the establishment of the cell lines, animals were anesthetized with isoflurane and euthanized by cervical dislocation, followed by the removal of the lungs. The lungs were mechanically dissociated, followed by chemical digestion (200 U/ml Collagenase type II, 200 U/mL Collagenase type IV, 1U/mL Dispase in DMEM) to create a single cell suspension. ECs were purified using the EasySep Mouse APC Positive Selection Kit (Stem Cell Technologies) after incubation with anti-CD31-APC antibody, as per manufacturer instructions. Cells were seeded at a density of 1 × 10⁵ cells/mL in Vasculife complete medium (Lifeline Cell Technology, Frederick, MD, USA). Cultures were incubated in humidified atmosphere with 5% CO₂ and 5% O₂. Cells were lifted after reaching approximately 80% confluency by incubation with 0.25% trypsin and counted using a hemocytometer. At least 3 distinct cell lines were used per group per experiment.