ROS and mito-ROS analysis

For total ROS and mito-ROS analysis, cells were harvested using trypsin digestion, or directly isolated from WAT. After washed with PBS, the cells were then resuspended with PBS containing 5 μM ROS indicator DCF (Invitrogen, C6827), or with 5 μM mito-ROS indicator MitoSOX (Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008). After staining for 30 min at 37°C in the dark, 2 volumes of ice-cold PBS were added to the cells, and the cells were kept on ice to terminate staining. The cells were then washed with ice-cold PBS twice and analyzed with flow cytometry. The data were analyzed with the FCS Express V3 software.