Hi everyone, I'm culturing mES recently, and I have found that my mES differentiate quite quickly, often on the second passage off MEF feeder. Could anyone with experience in preventing mES differentiation?
PS:I use MEF feeder plus 0.2% gelatin to culture mES. The medium used is Gibco's KnockOut DMEM, supplemented with Gibco's FBS, P/S，NEE amino acids， β-ME and LIF.
And the pictures of my mES are also enclosed
Hi Siyuan,
If you have obtained the cells from someone else, they may already contain differentiated cells, because I can still see good-looking, typical mES colonies in your culture. In this case, i suggest you to purify the cells based on differential adhesion: trypsinize the cells, transfer them to a gelatinized tissue-culture dish (10 cm2 dish), and wait for about 1 hour. Differentiated cells would attach to the dish very rapidly, while the undifferentiated ones remain suspended. Harvest the supernatant and transfer it to a new gelatinized dish (containing complete mES culture media) and you should normally see a largely undifferentiated culture after 1 or 2 days.
Alternatively, culture your cells in ground-state conditions (2i or R2i cultures). You can even culture the cells feeder-free.
Hope this helps.
Sharif
https://www.ncbi.nlm.nih.gov/pubmed/24036899
