Mitochondrial ROS production and mitochondrial content measurement

Mitochondrial ROS analysis was performed using the dye MitoSOX ({"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008, Invitrogen). Mitochondrial content was assayed using the dye MitoTracker FM ({"type":"entrez-nucleotide","attrs":{"text":"M22425","term_id":"197105","term_text":"M22425"}}M22425, Invitrogen), which passively diffuses across the plasma membrane and accumulates in active mitochondria.

20x10⁴ fibroblasts per condition were grown for two days, reaching 70% confluence in p100 plates. Cells were detached using trypsin for 5 min at 37 °C. For MitoSOX staining, cells were washed once using warm HBSS, incubated with 5 μM of MitoSOX in HBSS for 30 min at 37 °C, washed 3x using warm HBSS and suspended in HBSS. For MitoTracker staining, cells were washed with PBS, incubated with 0.2 μM MitoTracker for 30 min at 37 °C, washed 3x using warm PBS and suspended in PBS. Cells were directly analyzed via flow cytometry. In FSC and SSC, we first gated the population; next, two gates were set on SSC-A vs. SSC-H and SSC-A vs. SSC-W to exclude doublets. Based on an unstained control, MitoSOX+ and MitoTracker+ gates were set. Mean fluorescence of MitoSOX+ was normalized as a mean fluorescence of MitoTracker values, which represents ROS production per mitochondria. Antimycin was used as a positive control and FCCP as a negative control.