Mitochondrial-associated ROS levels and total intracellular ROS levels were measured by staining cells with MitoSOX and H2DCFDA. For mitochondrial superoxide, cells were washed with PBS solution, trypsinized and resuspended in PBS in the presence of 5 μM MitoSOX (Invitrogen) for 15 min at 37 °C. Subsequently, cells were washed twice with PBS solution containing 10% FBS and analysed by FACS using an LSRII instrument (Becton-Dickinson). For total intracellular ROS, cells were washed with PBS solution and incubated in HBSS solution in the presence of 5 μM H2DCFDA (Invitrogen) for 30 min at 37 °C. Afterwards, the HBSS solution was replaced with regular media and cells were incubated for an additional 1 h at 37 °C. Lastly, cells were washed with PBS solution, trypsinized and resuspended in PBS solution containing 10% FBS for FACS analysis using an LSRII instrument (Becton-Dickinson). As a positive control, cells were incubated in the presence of the mitochondrial inhibitor Antimycin A (Sigma) at a concentration of 0.1 μM for 30 min at 37 °C to induce ROS production. Each condition was done in two technical replicates with three biological replicates (n=6).
