This is a standard freezing protocol for freezing ES cells that can be used to freeze down vials of ES cells for future targeting or targeted clones.
1. Feed cells 2 hours before freezing.
2. Aspirate media, and wash the wells with PBS.
3. Add trypsin (0.05%) and incubate for 5 min at 37 C: 96-well plate - 30 ul/well
24-well plate - 100 ul/well
6-well plate - 500 ul/well
10 cm dish - 2 ml/plate
4. Add ES cell media at twice the volume of trypsin i.e. for 500 ul of trypsin, add 1 ml media
5. Titrate 4-5X
6. Place in a 15-ml conical and spin for 5 min at 1000 rpms
7. Make freezing media: 20% DMSO, 80% FBS.
For each vial of cells you will need 250-ul cold FBS and 250ul cold freezing media, for a total volume of 500 uls. For 1 confluent well of a 6-well plate we freeze down 4 vials/well.
To freeze 1 well of a 6 well plate:
FBS: 4vials X 250ul/vial = 1ml
Freezing Media: 4vials X 250ul/vial = 1ml X .2 = 200ul DMSO
X .8 = 800ul FBS
8. Have labeled tubes with loosened caps and media kept on ice.
9. Aspirate off supernatant of your 15-ml conical tube.
10. Add appropriate volume of FBS (only) and titrate.
11.
Add appropriate volume of freezing media, pipette 2-3X to mix and aliquot 0.5 ml/cryotube.
i.e. for a confluent well of a 6-well plate you would add 1 ml of FBS, titerate, add 1ml of freezing media, mix, and aliquot into 4 cryotubes.
12. Place cryotubes into a styrofoam tube rack and put in -80C. Cells can be placed into liquid N2 the next day.
NOTE: It is best not to leave cells longer than 2 weeks at -80 C.
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