It is important when making MEFs to use freshly thawed trypsin.
Use 0.05% Trypsin/EDTA from Gibco/Life Technologies, Cat. No. 25300-0054
1. Set up matings. If you are trying to produce Neo, Puro, or other drug resistant feeders use mice with appropriate genotype.
2. Check for plugs.
3. At day 13.5 dissect out uterus with embryos into sterile PBS into a 10 cm dish.
4. Under the hood, cut embryos out of the uterus and place in 2nd 10 cm dish in sterile PBS.
5. Cut off limbs, tail, and head and remove the internal organs. At this stage it is mostly the liver, which is a dark red spot.
6. Transfer the carcasses to a 50-ml tube and wash 3X in sterile PBS. Pipette to break up carcasses.
7. Transfer the carcass to a10 cm dish and mince with a sterile razor.
8. Add 5 mls of trypsin and transfer tissue slurry into a15 ml tube.
9. Place tube in 37 C H2O bath for ~ 1 min. Then rotate tube in hand for 2-3 minutes. Pipette the slurry 10X and then let the undigested tissue settle to the bottom of the tube.
10.Transfer the supernatant (containing the dissociated cells) into a 50 ml tube containing 25 mls of feeder media. Keep on ice.
11. Add 5 mls of fresh trypsin and repeat the digestion 2-3X. When there is only insoluble cartilage left the tissue will not settle down, at this point discard.
12. Combine the cells in the 50 ml tube by inversion and let the larger pieces settle down, around 1 min.
13. Plate the dissociated cells onto 10 cm plates. (2 plates for each embryo harvested)
14. Replace the media the next day. Continue to feed every two days.
15. Split 1:3 twice when the cultures becomes confluent.
16. Freeze down P2 culture just as it reaches confluence. Aliquot two 10 cm plates per cryotube.
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