Sp5 upright 2 photon confocal microscope

Cells were grown on Lab-Tek II Chamber Coverglass (Thermo Fisher Scientific, #155382) and incubated with 250 nM MitoTracker Red CMXRos (Thermo Fisher Scientific, # M7512) under routine culture condition for 30 min. Cells were then washed twice with PBS, fixed in 4% PFA/PBS and stained with DAPI at room temperature. Mitochondrial morphology was visualized by the Leica SP5 upright 2-photon confocal microscope. Relative intensities of Mitotracker were quantified in each cell using ImageJ as previously described (Ma et al., 2019 (link)). Briefly, confocal images were converted into binary images, and threshold was set to remove the cytosolic or background signals. Fluorescence intensity per cell was then corrected by subtracting the background signals. Unpaired two-tailed Student’s t test was used for statistical analyses.

ESCs or embryos were incubated with 10 μM EdU under routine culture condition for 90 min. For EdU tracing experiments, naive hESCs were first incubated with 10 μM EdU for 2 hr (day 0). After washing once with PBS, cells were cultured and harvested at day 2 and 4 for subsequent analyses. EdU-labeled samples were fixed and stained using the Click-iT^™ Plus EdU Flow Cytometry Assay Kits (Thermo Fisher Scientific, #C10632 or #C10634) according to manufacturer’s instructions. Samples were analyzed by flow cytometry analysis (for cells) or a Leica SP5 upright 2-photon confocal microscope (for embryos). EdU incorporation was quantified by median fluorescence intensity using FlowJo v10.0.7r2 software or mean fluorescence intensity using ImageJ.