1) Freezing medium
|M2 medium||6.5 ml|
2) Thawing medium
0.25 M sucrose in M2 (860 mg sucrose/10 ml M2). Prepare fresh and sterilize by filtering.
NOTE ! Depending on the strain 1-2 straws are thawed. Usually one straw is sufficient and contains 12-16 morulae for transfer to a pseudopregnant female.
ES cells should be split every other day 1:5-1:10. On the day of splitting the ES cell colonies may almost touch one another, but the plate should not be confluent.
ES cell medium is changed every other day and couple of hours before aggregation. For reagents of ES cell medium, contact the GM mouse unit.
For maintenance and freezing, ES cell culture on feeder cells is recommended
For aggregation, ES cells are cultured on gelatinized plates:
First aggregation in 24 hours: dilutions 1:3, 1:6, 1:12
Second aggregation in 72 hours: dilutions 1:6, 1:12, 1:24, 1:48
Note! Dilutions depend on the growth characteristics of your cell line. It is recommended to make several dilutions and pick the best for aggregation
SPLITTING OF ES-CELLS (6cm plate)
Take several ES cell plates and 50 ml of prepared ES cell medium to the GM mouse unit.
Note ! On the day of aggregation, the trypsinizing of ES cells to generate ES cell clumps is done in the GM mouse unit.