When I culture my mouse embryos at E 3.5, I let them stay on MEF coated dishes for 2-3 days, till they hatch and attach. I then trypsinize them and dissociate, later transfer to 96-well plates to culture for another 2-3 days. I hardly ever see any clones develop at this stage. Out of a dozen times or more, I have lesser than 50% efficiency of obtaining clones after transferring them to 96-well plates. Even if I do, I lose them in the next step, where I transfer them to 6-well plates. Could someone perhaps tell me in a descriptive manner how may I handle/ trypsinize or pipette my embryos, so I can improve my quantity of clones obtained.
Dear Pooja,
My questions are the follows:
1. What is the genetic background of your mouse embryos?
2. What kind of medium you use for ES establishment?
If you answer me these I can help you and maybe solve your problem.
Best,
Zsuzsa
Dear Zsuzsanna,
Thanks a lot for replying.
1.The embryos are derived from C57 BL/6 females crossed with CBA males.
2. I am using DMEM+non essential a.a+sodium pyruvate+Beta ME+PSQ (penicillin+streptomycin+L-Glutamine) added on use with Serum Replacement and LIF.
Hope it helps.
Thanks again.
Best regards,
Pooja
Dear Pooja,
The genetic background is not a problem. The establishment with F1 embryos are usually goes well. You can have difficulties with outbred stains such as CD1. The medium can be OK, but 15% KO serum and 2000 IU LIF are recommended. I will send you my ES est. protocol with deatels, this place is a bit short to explain it.
If the efficiency is still low you can try with 2i medium. I am sure it can increase your success.
All the best,
Zsuzsa
For ES cell establishment, I would recommend mechanical dissociation for the first few passages.
Dear Ron,
Thank you for your reply. Any specific reason why you would use only mechanical dissociation for the first passages? I do use mechanical dissociation, but along with Trypsin.
Thanks,
Pooja.
The reason simply is that I have found that survival and proliferation is better when using mechanical dissociation. It also allows for the selection and removal of the specific colony areas that have the desired morphology--that is, the ones that appear to be most like ES cells.
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