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Why is it recommended to thaw an entire vial of 5x10^6 cells into one well of a 6 well plate, but in the ES cell manual, it is not recommended to use more than 4x10^5 cells per cm2? (5 million cells in the well would be 5.2x10^6 cells per cm2).

The post thaw viability of the ES cells is about 80% (or about 4.6x10^6 cells). If you thaw the entire vial into a well of a 6 well plate (and the surface area in that well is 9.5 cm2), then that is about 4.8x10^6 cells per cm2. This is only slightly higher than the maximum recommended 4x10^6 cells per cm2. For some of the ES cells, that would be too many, but for others, just right. It was decided to err on the side of using too many cells because they can always be split.

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