We've been attempting to thaw and expand E14 mESC in feeder free conditions. We bought new GMEM, LIF, but are still having issues with the culture. Our protocol is as follows:
Plate dishes with 1% bovine gelatin.
Thaw cells as normal into mESC media (1x GMEM, 2mM glutamine, 1mM sodium pyruvate, 1x NEAA, 10% FBS (characterized, hyclone), 1:1000 beta mercaptoethanol, 1000 units LIF (ESG1107)
Thanks for your help!
Hi Kelly,
Have you considered using 2i instead of Serum/LIF?
Best
Bastian
I hadn't, Bastian, thank you for the suggestion.
It looks like a combination of 2i and 2% serum produced happy adherent colonies -
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081156
If you want to exclude serum all together you also just go with the original formulation published by the Smith lab back in 2008
http://www.nature.com/nature/journal/v453/n7194/abs/nature06968.html
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