Egm bulletkit medium

Tubastatin A (Calbiochem, San Diego, CA) was diluted in phosphate buffered
saline (PBS) at a stock concentration of 500 μM, aliquoted and frozen for later
experiments. Human umbilical vein endothelial cells (HUVEC) (CC-2519, Lonza, Basel,
Switzerland) were grown in EGM BulletKit medium (Lonza, Basel) in a humidified atmosphere
of 95% air and 5% CO₂. Cells were used for experiments from
passage 3-8. After cells had reached 70% confluency, medium was exchanged with
glucose-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
0.5% fetal bovine serum (FBS), with and without 15 μM TubA. Cells
subjected to normoxic conditions were placed in a humidified chamber with 95% air
and 5% CO₂. For anoxic treatment, HUVECs were placed in a MIC-101
modular incubator chamber (Billups-Rothenburg, Del Mar, CA, USA), which was flushed with
5% CO₂ and 95% N₂ for 15 minutes, and maintained in
a 37°C environment for 24-48 hours. The anoxic conditions for final testing were
selected after a series of pilot experiments. Our goal was to stress the cells without a
major impact on their actual viability, as proposed by Lee et al(32 (link)).
The study groups were: (1) normoxia, (2) normoxia plus 15 μM TubA, (3)
anoxia, and (4) anoxia plus 15 μM TubA.

Murine pancreatic endothelial MS-1 cells and peritoneal RAW 264.7 macrophages (ATCC, Manassas, VA, USA) were grown in DMEM with 10% low endotoxin fetal bovine serum (FBS; PAA, Pasching, Austria) and 1% Penicillin/Streptomycin. Human umbilical vein endothelial cells (HUVEC) from Lonza [26 ] were cultivated in EGM Bullet Kit medium (Lonza).
ECs were treated with 1.0 μM OA-NO₂ with or without cytokines – IFN-γ (50 ng/ml), IL-1β (5 ng/ml), TNF-α (10 ng/ml), or TGF-β (10 ng/ml) – for different time periods (20 min - 48 h). The inflammatory phenotype in macrophages was induced using bacterial lipopolysaccharide (LPS, 100 ng/ml) treated for 24 h. Before each experiment, ECs and macrophages were cultured in complete media as indicated above. Two hours before the start of experiments, the complete medium was replaced with DMEM with 2% of FBS or FBS-free DMEM. In long-term experiments (6 d), medium and treatments were renewed regularly after 2 days. The final concentration of OA-NO₂ used for all experiments (1.0 μM) was selected on the basis of its physiological relevance and our previous experience [2 , 20 (link)]. OA-NO₂ was applied alone or together with cytokines. Cell viability was measured by ATP Cell Viability test (BioThema, Handen, Sweden) [27 (link)]; no effect of cytokines or OA-NO₂ exposure was detected (data not shown).