Endothelial Cell Treatment with Antioxidants

Human dermal microvascular endothelial cells, HMEC-1 (purchased from ATCC, Cat. No. CRL3243™), were cultured in MCDB 131 Medium (Thermo Fisher Scientific) supplemented with FBS (10%, Thermo Fisher Scientific), L-glutamine (2 mM, Thermo Fisher Scientific), hydrocortisone (0.05 mg/ml, Sigma-Aldrich), Epidermal Growth Factor (5 ng/ml), 100 U/ml penicillin, 10 μg/ml streptomycin, and 250 ng/ml amphotericin B (Sigma-Aldrich). HMEC-1 were cultured under standard conditions (37°C, 5% CO₂) and passaged two times a week. In all experiments, cells between the second and tenth passages were used only when they reached full postplating confluency. Bardoxolone methyl (CDDO methyl ester, Cayman Chemical), dimethyl fumarate (Sigma-Aldrich), and L-sulforaphane (Sigma-Aldrich) initially diluted in DMSO were added to the culture medium at final concentrations of 100 nM, 300 nM, 500 nM, 1 μM, 3 μM, and 5 μM in triplicates, if not stated otherwise. Control cells were treated with 0.05% DMSO added to the culture medium. To check for acute toxicity, the cells were incubated with tested agents for 3 hours. Standard toxicity assessment time was set to 24 hours, according to the literature [30 (link)].