Metamorph v 6

Intracellular ROS levels were quantified by dihydroethidium (DHE), which is oxidized by superoxide to yield ethidium; the latter binds nuclear DNA and emits a red fluorescence (535 nm Ex; 610 nm Em). The substrates NADH (1 mM), succinate (5 mM), l-arginine (1 mM), and xanthine (1 mM) were added to identify ROS sources, along with the inhibitors: DPI (Diphenyleneiodonium) (0.1 mM) to inhibit NADPH (nicotinamide adenine dinucleotide phosphate) oxidase; antimycin (0.05 mM) for mitochondrial complex II; l-NAME (N(ω)-nitro-l-arginine methyl ester) (1 mM) for eNOS; and allopurinol (0.02 mM) for xanthine oxidase.
Fifteen minutes later, the cells were incubated with DHE 20 µM and washed three times with warm PBS. Then, the cells were placed in fresh medium, analyzed, and photographed in a Cytation 5 cell imagen multimodal plate reader (Biotek Instruments, Winooski, VT, USA), using the Gen 5 software (Biotek Instruments) under a 20× objective. Finally, the intensity of DHE emission was quantified with Metamorph v. 6.1 (Molecular Devices, San Jose, CA, USA).

After 4 h of cell seeding, cell migration studies were started and carried on for 6 h. Each cell was tracked until cell division occurred. Hence, we restricted our cell migration study to 6 h. The study focused on early intervals following seeding since the cell density is low during this period, helping us avoid impact of cell-cell interactions. Six representative areas of each substrate were selected for acquiring bright field images. Cells were tracked based on their centroid. Imaging was carried out every 15 min. Time-lapse videos were analyzed with METAMORPH (v. 6.1, Molecular Devices, Sunnyvale, CA, USA) in order to extract cell trajectories. Mean migration rate was defined as the total movement of the call per unit time and directionality was assessed as movement across vs movement along pattern direction^{41 (link)}. Mean migration was averaged for six cells on each type of substrate.