21. In general cells should be replated on the appropriate size plate (96- or 24-well plate when passaging from 96-well plate, 6-cm dish when passaging from a cryovial), and grown up so that there are enough for freezing into cryovials and introducing into mice.
22. A separate plate should be prepared for aggregation as was described in the protocol. The reason for highly diluted single-cell plating is to produce clumps of 10-25 loosely connected cells prior to aggregation. Then the required-size colonies are predominantly found on the plate. Care should also be taken in order not to disaggregate the cell clumps (by pipeting the cells too vigorously or overtrypsinization).
23. For blastocyst injection purposes, cells do not need to be maintained as clumps. Therefore they should be completely dissaggregated.
Was this article helpful?