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Please help, may i replenish Glutamine in cell culture medium? - (Apr/20/2012 )

Hallo, I am new in cell culture
I hope that somebody here helps me with my problem.
I have cell culture media 6 litres alpha MEM suplemented with 2mM glutamine from manufacturer. I store them in the fridge. I will obtain my cell line in July and then I can start my work in cell culture.In this long time period my Glutamin in media degrades in ammonia, I think. Could I replenish it with a solution of Glutamin (stored at -20C) just before use of the media, or is it not correct?
I can not buy new media without glutamin or with its stabile form (L-Ala-L-Glu).
Please give me a advice.


Dear Didiana,

I have ordered Glutamine containing media in th past and kept it well past it's use by date......years after. I have then used it and further supplemented it with L-Glutamine ....and seen no problems with:

Cell growth
Oxygen consumption rates
Inducibility of enzymes

In fact all the cellular parameters we have checked have been the same as with media that is in date

Hope this reassures you

Kindest regards

Uncle Rhombus


Hi Didiana,

As Rhombus says, you can just supplement with L-Glutamine.
In fact, I often supplement L-glutamine in brand new media, and it does not effect my cell cultures, although I would not advise you to do so unless you know it doesn't affect your cells.




Dear Rhombus,

thank you very much for your message, so then I will replenish all my media with 2mM glutamin before I use them for subculturing and experiments. And what about the toxicity of AMMONIA, does not it attack my cells negatively?