Differentiation problem despite new LIF - (Jun/04/2006 )
hi,
i work with mouse ES cells. I was finding that the ES cells (R1 and D3 lines) started to differentiate 2-3 passages after thawing from stocks. I thought it was LIF so i shifted to new LIF.The problem continued-so i changed the serum(as it was not batch tested.). i started using knockout serum replacement. the problem seems to have continued.
Which of the following things have an effect:
1) trypsin-i am using 0.25%-should i use lower percentage?
2) i am not supplementing glutamine- i use gibco DMEM which comes with glutamine added.
is there anything else which might cause this problem?
i work with mouse ES cells. I was finding that the ES cells (R1 and D3 lines) started to differentiate 2-3 passages after thawing from stocks. I thought it was LIF so i shifted to new LIF.The problem continued-so i changed the serum(as it was not batch tested.). i started using knockout serum replacement. the problem seems to have continued.
Which of the following things have an effect:
1) trypsin-i am using 0.25%-should i use lower percentage?
2) i am not supplementing glutamine- i use gibco DMEM which comes with glutamine added.
is there anything else which might cause this problem?
What else is in your medium? Are you adding B Mercoptoethanol?
Your Trypsin is 10X the concentration I use and mine is supplemented by 1% Chick serum and 1mM EDTA .
I dont think the Glutamine is the problem.
Hope this helps
How often do you pass them? Confluency is a big factor. Are you plating them dense enough? They like to have plenty of neighbors, but not too crowded. I like to pass them somewhere around 70 - 80 % confluency. Usually every other day. If I let them go the whole weekend, I have trouble with differentiation.
Have you ever think of mycolplasma？in my experience，when your ESCs were contaminated with mycoplasma，it always shows a tendency of differention and lower proliferation，lower ratio of nuclei to cytoplasm。you may try to use some anti－mycoplasma reagent，good luck！