Measurement of cell ROS/Superoxide production
For the evaluation of oxidative stress in live cells, the ROS-ID Total ROS/Superoxide Detection Kit (Enzo Life Sciences, Inc), was used according to the manufacturer’s instructions. Briefly, after cell treatments for 48h, cells were incubated with reactive oxygen/nitrogen (ROS/RNS) detection reagent and Superoxide detection reagent both used at a final concentration of 3 μM in wash buffer for 1h in the dark in a humidified 37 °C incubator. After incubation, chips were washed with wash buffer and immediately observed. For positive controls, chips were simultaneously incubated with the ROS Inducer (Pyocyanin) in wash buffer at 500 μM, and with the detection probes for 30 min. at 37 °C in the dark. Chips were observed under a confocal microscope (A1R MP Eclipse Ti-2, Nikon) and MFIs of the ROIs occupied by the cells were measuredvia an automated acquisition/analysis software (NIS Elements AR, Nikon). ROS/RNS were detected using a 488 nm laser line with a FITC filter set (525/50 nm band-pass, BP), while Superoxides were detected using a 561 nm laser line with a TRITC filter set (595/50 BP). MFI levels were subtracted for endogenous fluorescence of untreated cells. Results were plotted as the ratio between each treatment and its own internal control.