I am using Mouse (ICR) Inactivated Embryonic Fibroblasts (Thermofisher, Cat No:A24903) to culture ES-E14TG2a Cell Line ( Sigma, Cat No: 08021401-1VL). It is suggested that approximately 1.5*10^6 MEF cells should be plated to culture mESC cells in T25 flask. If I increase density of MEF cells to 4*10^6 per T25 flask during culturing of mESC, Do I encounter any problem such as differentiation of mESC, low MEF depletion efficiency during MEF Depletion etc.?
We use MEF to provide Leukemia Inhibtory Factor (LIF) for mESC. High number of MEF means that more LIF for mESCs. Why do high number of MEF cause differentiation of mESCs?
Hello
Yes this may affect of your results
See the protocols and tips here .
https://tools.thermofisher.com/content/sfs/manuals/feeder_dependent_mESCs_in_ksr_lif.pdf
https://catalog.coriell.org/0/sections/collections/nia/nia_protocol_mesc.pdf
If using feeders, you should stick to the recommanded density. Feeders are there for LIF and other secreted factors, for providing a suitable matrix (that means not too many and not much). They should not exhaust the nutriments in detriment of the ESC.
Why would you change the feder concentration?
PS: for ES-E14TG2a, you can remove completely the feeders and growth only on gelatin coated plates. but then you need to have 2i in your medium.
- Badges
- Science topic