Day -1 Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells. 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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