Quantification of mitochondrial reactive oxygen species in living cells by using multi-laser polychromatic flow cytometry

Reactive oxygen species (ROS) are constantly produced in cells, mainly by mitochondria, as a consequence of aerobic respiration. Most ROS derive from superoxide, which is rapidly converted to hydrogen peroxide. ROS are involved in the regulation of several physiological and pathological processes, and the possibility to measure them simultaneously is needed, when the redox status of the cells is modified by experimental/biological conditions. Flow cytometry is the main technology that generates multiple information at the single cell level in a high-throughput manner, and gives rapid and quantitative measurements of different ROS with high sensitivity and reproducibility. Here, we describe a novel approach to detect simultaneously mitochondrial hydrogen peroxide and mitochondrial superoxide in living cells. The staining has been performed by using the fluorescent dyes MitoSOX Red Mitochondrial Superoxide Indicator, Mitochondria Peroxy Yellow 1, Annexin-V Pacific Blue conjugate, TO-PRO-3 iodide, anti-CD4-APC-Cy7 and -CD8-Pacific Orange mAbs. We used this approach to quantify mitochondrial ROS in CD4+ and CD8+ T cells form patients affected by Down syndrome and age- and sex-matched healthy donors.