Generation and imaging pipeline for aggregates of mESCs (ICM organoids). Mouse tet::GATA4 ESCs were pre-cultured for 3 days in medium containing serum and LIF (S + L) and PD0325901 (PD03). At day 3, cells were stimulated with dox for 6 h to induce PrE differentiation. After dox removal, 200 cells were seeded in microwell plates coated with 1% low melt agarose. Aggregates were formed in medium containing S + L and were kept undisturbed. After 24, 48, and 72 h, aggregates were collected, stained, mounted, and imaged. To see this figure in color, go online.