Opterra 2 multipoint confocal system

Prior to time‐lapse analysis, 3000 ESCs (Zscan4⁻ and 2C::tbGFP⁻) were FACS‐sorted into individual wells of a gelatin‐coated glass bottom 96‐well plate (ThermoFisher) containing 50 μl of ES cell media. Afterwards, ES cell media containing sodium acetate, sodium lactate or no added metabolites were added to each well, to a final concentration of 32 mM and a final volume of 150 μl. Cells were then allowed to attach for a couple hours. Image acquisition was carried out in four positions within each well with a 20 × 0.75 NA Plan‐Apochromat objective lens every 30 min for 96 h using a Nikon Ti‐E system equipped with the Bruker Opterra II multipoint confocal system. Images were recorded on an EMCCD camera using emission filters for turboGFP (BP520/40), mCherry (570LP) and iRFP (655LP) mounted on a FLI filter wheel. Spontaneously arising Zscan4⁺ or 2‐cell‐like cells were manually identified using the ImageJ software and quantified relative to the total number of cells present in the field of view at each specific timepoint. Time‐lapse experiments were carried out in three independent biological replicates.