Freezing Plates
Materials and Reagents
- Dry Ice
- EmbryoMax ES Cell Qualified Freezing Medium — Millipore cat. no. ES-002-D
- ES Cell Medium
- Parafilm® Film
- Pipette
- 0.05% Trypsin-0.53mM EDTA — Millipore cat. no. SM-2002-C
Protocol
- When the ES cell colonies have regrown, wash with DPBS and add 35 µL of Trypsin. Incubate for 10 minutes at 37°C, and then add 65 µL of ES Cell Medium.
- Disperse the cells with a pipette and transfer into a replica plate containing 65 µL of cold EmbryoMax ES Cell Qualified Freezing Medium (2x).
- Wrap each plate with Parafilm and place on dry ice for 20 minutes. Transfer to –80°C to freeze. Plates can keep for a number of months.
- To thaw, add 150 µL of ES Cell Medium plus selection agent to each well. Thaw plate quickly by placing in a 37°C incubator.
- Transfer the thawed plates to a hood and manually pipette each well. Transfer the contents to a fresh 24-well plate with PMEF feeders (if required).
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Pathways
