Live tracking of Gata6 reporter expression

To track Gata6 reporter expression in live cells, PrE-like differentiation was induced by a 6 h pulse of doxycycline-treatment in serum-containing medium as described in (Schröter et al., 2015 (link)). Then, 16 h after doxycycline removal, cells were either switched directly to N2B27 medium lacking Phenol Red or trypsinized, sorted for reporter expression and seeded on fibronectin-coated imaging dishes (ibidi µ-slides). Time-lapse imaging was started within 2 h after sorting on an Olympus IX81 widefield microscope equipped with LED illumination (pE4000, CoolLED) and a Hamamatsu c9100-13 EMCCD camera. Hardware was controlled by MicroManager software (Edelstein et al., 2001 ). Time-lapse movies were acquired using a 40× oil immersion lens (NA 1.2), with 10 min time intervals.
Cell tracking was carried out with TrackMate (ImageJ) (Tinevez et al., 2017 (link)) based on the constitutively expressed H2B-Cerulean nuclear marker. Fluorescence intensity was measured in a circular region of interest in the center of the nucleus, and background-subtracted fluorescence intensities plotted in Python. Trace color in Fig. 6D was assigned according to fluorescence intensity in the last frame of the movie, with respect to the estimated intensity threshold used for flow sorting (dashed line in Fig. 6D).