When I culture cells in N2B27-based medium, they adhere very poorly to surfaces, irrespective of the presence or absence of the 2i inhibitors. I want to do some experiments with 2i cells which require that they adhere to a surface. Has anyone seen any research in which gene expression has been studied in cells that have been cultured in serum/2i/LIF?
Many thanks,
Oliver
Hi Oliver,
PD and Chiron are working also in serum of course. I have more experience with Chiron though but I've heard of labs using both inhibitors to maintain recalcitrant mESCs in culture even if I never heard of any publication using Serum2iLif. However I would not culture cells this way, reviewers are going to complain about that and serum contains growth factors that will partially interfere with the outcome of the two inhibitors usage. One thing you can do is to add a small percentage of BSA or Serum (below 5% if I recall correctly) to 2iLif (in N2B27). This will help cells to attach, it is quite accepted and there should be also some publication about that, I'll look into it when I have some time if you need it.
Hope it helps
Ah that's great Francesco, thanks for that. I do have a small amount of fraction V albumin in my medium, but it doesn't seem to make much difference vis-a-vis adhesion and colony morphology. If you do have a minute to find a specific reference for adding small amounts of serum, that would be really helpful. I can heat-inactivate serum to denature the growth factors, if that helps things... Thanks again!
Hi Oliver,
there should be at least another one, but you can take a look at this to start with:
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0081156
1/2 % of serum should be sufficient, I didn't have time to read the outcome of BSA usage.
Best
Francesco
Thanks for that! It's such a tricky topic to search for - search terms like "2i and serum" turn up thousands of papers that are comparing the two "cell types". A classic needle in haystack problem. Most of the research heavily confounds the effects of serum and the effects of the inhibitors, too. I see a lot of the morphological changes ascribed to 2i when I switch to serum replacement, even without the inhibitors present...
I never try to add 2i in medium containing serum. I however I sometime cultive mESC in Knock out serum remplacement medium + 2 inhibitors. I works very well, the cells are still able to transmit to the germline when injected into blasocysts.
and yes, we do see too a lot of morphological changes when we change serum for Knock out serum remplacement or vice versa.
The "Knock out serum replacement medium + 2 inhibitors" sounds very close to the standard recipe for 2i medium - you would just add the N2 and B27 supplements to that. I don't have the facility to make chimeras, but I wonder how the efficiency of germline transmission is influenced by 2i/serum vs 2i/serum replacement... might be a good thing for someone else to look at!
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