To evaluate superoxide production by mitochondria, the MitoSOXTM Red reagent (1.25 μM, Molecular Probes) was used in live cells, a fluorogenic dye specifically targeted to mitochondria in live cells. This reagent is readily oxidized by superoxide, but not by other ROS- or reactive nitrogen species (RNS)-generating systems. Oxidation of MitoSOXTM Red reagent by superoxide produces red fluorescence. For these experiments, astrocytes were incubated for 24 h with different treatments; ethanol (10, 50 mM), LPS (50 ng/ml), ATP (5 mM) or rotenone (25 μM) as the positive controls, and with inhibitors Z-YVAD-FMK (Calbiochem) or NLRP3 bp or Mito-TEMPO. Then cells were further incubated with MitoSOXTM (1.25 μM) for 30 min at 25°C in the darkness. Cell samples were acquired with a Cytomics FC500 flow cytometer (Beckman-Coulter, USA) equipped with 488 and 635 nm lasers detectors for forward scatter (FS) and side scatter (SS). We defined the cell population with the FS and SS parameters, MitoSOX Red was excited by laser at 488 nm, and its fluorescence emission was collected at 620 nm. In order to perform the analysis in live cells,
