*4.11. Intracellular ROS Detection*

Intracellular ROS formation was evaluated in Caco-2 cells treated in the different experimental conditions using a 2- ,7- -dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe [20]. Cells were incubated for 45 min with pre-warmed PBS containing the fluorescent probe (25 μM). After incubation in the dark at 37 ◦C, cells were washed

twice in PBS. Fluorescence of labeled cells was measured in a fluorescence plate reader at λex/λem (485/535 nm) (Multi-Mode Microplate Reader SynergyTM HT, BioTek Instruments, Inc.). Data obtained in cells treated in different experimental condition were normalized to the results of the cell number. Data are expressed as arbitrary units of fluorescence (A.U.). An automated microscope (Lion hearth FX Automated live cell Imager, Biotek, BioTek Instruments, Inc., Santa Clara, CA, USA) was used for cellular imaging.
