Fig. 4.

Application and validation of multiplex ROS-cell viability assay for HT and LT application on angiogenesis-on-a-chip. Micro-vessels of HUVECs cultured against ECM under perfusion and stimulated to generate sprouts were treated with TBHP 10 mM with and without curcumin 67 μM, standard media or 0.1% of DMSO and stained for DNA with Hoechst (blue), for ROS with DHR123 (green) and dead cells with PI (red) (A) Representative images of max-projection of entire micro-vessel (B) ROS content in a log scale expressed as fluorescence intensity of DHR123 normalized against the number of cells count (C) Cell viability expressed as percentage (D) LT application on angiogenesis-on-a-chip: representative images of max-projection of entire Z-stack of quiescent (micro-vessel) and angiogenic (sprouts) ECs. Zoom-in on the angiogenic part of the angiogenesis-on-a-chip model treated with 10 mM of TBHPTBHP and 67 μM of curcumin (E) ROS content in angiogenic and quiescent ECs, expressed as ratio between ROS fluorescence intensity and nuclei count in a log scale. Data are expressed as average ± STD and in terms of replicates graph in (B) and (C) N = 3 and n = 2–7 while graph in (E) N = 1 and n = 3–7. Difference between results in graph (B) and (C) were evaluated by one-way ANOVA with Tukey's multiple comparison P-value < 0.05 was considered statistically significant * p = 0.0245, **p = 0.0014, ****p < 0.0001. While in graph (E) two-way ANOVA with Sidak's test was used to compare ROS content in angiogenic and quiescent ECs, p-value < 0.05 was considered statistically significant **p = 0.0024, *** = p = 0.0009 ****p < 0.0001. All scale bars are 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)