After completing an experiment I've found that the medium used for differentiating mESCs (spontaneous) was with excess of L-Glutamine content. In what way does this affect the spontaneous differentiation ? Can I still consider my results valid or should I repeat my experiment with normal level of Glutamine ?
Here's the absolute answer: You need to report your results in the context of your methods, so that others can reproduce them. So you need to say what the [gln] was: whether "usual" or unusual.
The problem is that no-one really does that. They say "DMEM" and infer that includes 0.584 g/l glutamine. They don't specify how old the medium was or whether they added more, etc.
Here's some important information:
Glutamine is unstable and its half life depends on the storage conditions of the medium, and the type of medium itself (and the amount of bicaronate or phosphate inthe medium).
Medium can be purchased with or without gln. I prefer to get medium without Gln, and to add it just before use. That way I know what I have. But different labs have different protocols, such as re-adding gln every two weeks or month. This despite the halflife is reported to be about one year, so there is an accumulation of gln!
Here is a web tool with lots of basic culture medium information especially about components:
http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert.html
Now to basics:
How much of an excess did you add? What did you add it to? HOw old was the medium and how much gln did it have to begin with?
I am not aware of any studies sytematically looking at the effects of [gln] on mouse ES cell differentiation.
For all of that either repeat the experiments with "normal" glutamine, or report the amount you used (and risk others scratching their heads wondering why you used such an odd amount!
Ian,
thank you for the information and clarification. Double the amount of glutamine was added to a fresh medium.
In principle due to the instability of Gln you are never sure on its effective concentration during the experiment. I agree with Ian about the best choice being repeating the experiment, if you cannot repeat it keep track of the results and correlate to the double gln used. I don't know the effect on the differentiation, it may alter the metabolic activity.
The easiest way to eliminate this complication is to supplement your media with GlutaMAX rather than glutamine. With the glutamine as part of a dipeptide, it is stable but still metabolically available.
mESCs depend on LIF for maintaining their pluripotency. In LIF-deprived culture, cells start undergoing differentiation, which is spontaneous until any factors that induce specific differentiation are not added.
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