Passaging ES Cells

11. The cell pellet should be carefully resuspended. If this is not carried out, the ES cells will grow as large clumps, containing necrotic centers and differentiated cells at the periphery.

12. ES cells are growing optimally if they are ready for passing on each alternate day, and if their morphology does not change during this time. The typical ES cell morphology is when single cells are not visible, and they grow as characteristically shaped small clumps.

13. The number of cells to be plated at each passage and length of growth between two passages is critical. Cells should not be left to overgrow, and care should be taken not to split cells too diluted or too dense.

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