MTT Assay and Cell Count

HUVEC and HDMEC were seeded in 96-well plates and exposed 24 h later to different doses of CYT D. After 24 and 96 h (HUVEC) or 24 and 72 h (HDMEC) of treatment, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/ml; Sigma-Aldrich) was added in the culture medium in a ratio of 1:10. After 4 h of incubation with the MTT solution, formazan crystals, derived by the degradation of MTT into the mitochondria of living cells, were dissolved in ethanol/DMSO (1:1), and absorbance was measured at 550 nm using Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific). Viability was calculated by comparing the absorbance of each treatment to the untreated (-) sample at 24 h. The experiments were repeated five times in triplicate.

For samples deriving from the RWV, the beads were collected from the vessels, washed with phosphate-buffered saline, trypsinized, stained with trypan blue solution (0.4%), and counted using Luna Automated Cell Counter (Logos Biosystems, Anyang, South Korea). The experiment was performed three times in triplicate.