Flow Cytometry, Fluorescence Activated Cell Sorting, and TMRE Staining

Cells for flow cytometry analysis were dissociated into single cells by TrypLE (Thermo Fisher Scientific, #12604021) and resuspended with cold PBS buffer. BD LSRII or Beckman CytoFlex LX was used for flow cytometry analysis. Flowjo or CytExpert software were used for data analysis. Fluorescence activated cell sorting (FACS) was performed with BD Influx. For separation of GFP⁺dsred⁺ and GFP⁺dsred^– cells in Supplementary Figure S4, HA-GFP and HA-mitodsred cells were co-cultured for 48 h before FACS.

For TMRE staining, cells were washed with 0.2% FBS-containing DMEM/F12 for twice. Then cells were incubated with 500 nM TMRE (Thermo Fisher Scientific, #T669), which was diluted in 0.2% FBS-containing DMEM/F12, in the incubator for 30 min. After the incubation, cells were washed with 0.2% FBS-containing DMEM/F12 for twice and then dissociated into single cells with TrypLE. Beckman CytoFlex LX and CytExpert software were used to read and analysis TMRE intensity.