Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.
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Day 1: Trypsinized the cells as for normal passaging until the colonies lift off. Try to keep the loosely connected clumps of cells together by gentle handling. Then directly plate the cells 1:3 into bacterial grade Petri dishes in LIF free ES cell medium.
Day 3: Aspirate the medium carefully. Avoid sucking up too many of the aggregates. Then add new medium.
Day 5: Aspirate as in Day 3 and replace the medium. At this stage the differentiation the aggregates are called simple embryoid bodies.
Day 7: Change the medium
Day 9: Change the medium
Day 11: Change the medium
Day 13: Change the medium
Day 15: By this day a high percentage of the embryoid bodies will have developed fluid filled cavities. The structures are called cystic embryoid bodies
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