Gastruloid Generation and Immunofluorescence Analysis

Gastruloids were generated as previously described^{6 (link),9 (link),69 (link)}. Briefly, 300–700 mESCs were aggregated in 40 μl N2B27 in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning). After 48 h, 150 μl per well of 3 μM Chi in N2B27 were added. At 72 h, 150 μl of medium were removed and substituted with 150 μl of fresh N2B27. From 96 h, the medium was changed to N2B27+++ which contains 30 ng ml⁻¹ bFGF (PMG0034, Gibco), 5 ng ml⁻¹ VEGF 165 (PHC9394, Gibco) and 0.5 mM L-ascorbic acid phosphate (013-12061, Wako). From 120 h on, half of the medium was changed daily. From 144 h, N2B27 was used for daily medium changes. For immunofluorescence analysis, gastruloids at 96 h were transferred in Ultra-Low Attachment 24-Well Plates (3473, Corning) with 100 μl of medium, plus 700 μl of fresh N2B27+++, and cultured on an orbital shaker placed at 37 °C, 5% CO₂ at 100 rpm (VWR mini shaker), with the same culture schedule.