Quantifying Intracellular and Mitochondrial ROS

Intracellular and mitochondrial ROS analyses were performed as previously described [37 (link)]. Briefly, the levels of intracellular ROS were determined by incubating cells for 10′ at 37 °C with the redox-sensitive probe 2′-7′-Dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions of the manufacturer. CM-H2DCFDA fluorescence was analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences) and data were processed with FlowJo software (Tree Star, Inc.). After treatments, cells were incubated with 5 µM MitoSOX Red (MitoSOX Red Mitochondrial Superoxide Indicator, Thermo Fisher Scientific Inc.) for 10 min at 37 °C and then 2.0 × 10⁴ cells were acquired by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). Fluorescence data were processed with FlowJo software (Tree Star, Inc.).