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.