Quantifying Apoptosis in mESCs

To detect apoptosis of mESCs, Cleaved Caspase-3 (Cell Signaling, 94530) and 7-AAD (Thermo Fisher, 00-6993-50) were measured to assess the middle and late stage of apoptosis respectively. mESCs were seeded onto the micropatterns, ensuring complete coverage, and cultured for 16 h. Subsequently, the medium was changed to a LIF-free medium. To assess the middle stage of apoptosis after 48 h differentiation, fixed cells were stained with Caspase-3 (cleaved) as mentioned in the section of “ Immunostaining”. To assess the late stage of apoptosis after 48 h differentiation, live cells were stained with 7-AAD and Hoechst 33342 (Sigma-Aldrich, B2261-25MG) for 30 min at a 37 °C incubator. Finally, the cells were imaged using a spinning disk confocal microscope (OLYMPUS UPlanFL N 10x/0.30 N.A. objective and Andor iXon camera). Quantification of apoptosis from nuclear morphology was performed on DAPI images acquired by ARTseqFISH as previously described^{62 (link)}.