Anxvf 200t

Cells were preloaded with 100 nM MitoTracker^® Green FM (MTG, Thermo Fisher, M7514) and washed with phosphate-buffered saline (PBS) before treatment [19 (link)]. To monitor mitochondrial mass, cells were collected in FACS tubes and loaded with MTG, as previously reported [19 (link)]. For the assessment of cell death, cells were stained with annexin V-FITC (Immunostep, ANXVF-200T) for 15 min at 37 °C, and afterwards, propidium iodide (0.1 mg/mL) (PI, Sigma-Aldrich, P4170) was added to each tube to detect either the percentage of PI or annexin-positive (+) cells. Otherwise, we measured ROS levels by detecting the accumulation of superoxide with 5 μM dihydroethidium (Invitrogen, D1168). To study the mitochondrial membrane potential (MMP) and mitochondrial ROS, we used 20 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM, Invitrogen™, T668), and 2 μM MitoSOX^TM (Invitrogen™, M36008), respectively. MitoSOX and dihydroethidium were oxidized to ethidium and emitted a red fluorescence. All stained cells were analyzed by flow cytometer (Beckman Coulter FC500-MPL).

Cells were stained with annexin V-FITC (Immunostep, ANXVF-200T) in PBS for 15 min at 37 °C. Propidium iodide (PI, Sigma-Aldrich, P4170, 0.1 mg/mL) [20 (link),24 (link)] was subsequently added at the end of incubation to determine the percentage of cell death (apoptosis and/or necrosis) by flow cytometry (n = 10,000 events) (Beckman Coulter FC500-MPL).