Measurement of Cellular Oxidative Stress and Glutathione

Cells were washed with PBS (–) and treated with 5 μM CellROX Oxidative Stress Reagents (cat. no. C10444; Life Technologies, Carlsbad, CA, USA) for ROS staining, or 20 µM ThiolTracker Violet (cat. no. T10095, Molecular Probes, Eugene, OR, USA) for GSH staining.
Fluorescence images were acquired using an EVOS™ M5000 imaging system (Thermo Fisher Scientific Inc.). Fluorescence intensity was quantified using ImageJ software v1.52A (National Institutes of Health; http://imagej.nih.gov/ij/). For corrected total cell fluorescence (CTCF), we used the following formulas^{61 (link)}:

CTCF = integrated density (total area of selected cells × mean fluorescence of background readings)

CTCF per cell = CTCF/ Ncells

where “Integrated Density” is the integrated intensity of the pixels for all cells in the image, total cell area is the number of pixels of all of the cells, background fluorescence is the average mean gray value of nearby regions containing no cells, and Ncells is the number of cells that was measured by counting fluorescently labeled nuclei from images.