What additives can I add to the culture medium to improve the expansion of a Jurkat cell in a 96-wells plate?
I have tried to isolate a transduced single jukat cell without a selection marker.Then I expand it to a large number of jurkat cells. but during culture, jurkats become apoptotic and die after 3 weeks. why?
what can I do to have a clonal jurkat cell line from a single cell?
Hello
I would suggest you add conditioned medium of Jurkat culture to the culture medium in the ratio of 1 :1.
Presence of growth factors, cytokines and other growth promoting components in the conditioned medium will help in the growth of single cell.
Best.
Malcolm Nobre I have already tried to use conditional media. but it wasn't enough to Jurkat cells proliferate. I don't know why their growth stops after transduction!
Another reason why cells die at low density is that the cells leak small amino acids, such as glycine, alanine and serine. You can either add non-essential amino acids to your culture medium, but the simplest solution is to add 1 mM sodium pyruvate. This is a standard cell culture reagent.
Pyruvate is transaminated into alanine, which can be converted to glycine; it is converted into serine by a slightly more complicated pathway..
Gary Lee Gilmore thanks for your reply.
I use the powder for preparing of RPMI 1640, so it already has sodium pyruvate in its formulation. did you tell me to add it more?
Article High Efficiency Ex Vivo Cloning of Antigen-Specific Human Ef...
The methods in the article I linked above describe how the authors performed limiting dilution of human T cells.
The formulation of standard RPMI 1640 does not contain pyruvate, but it does contain all amino acids, so cells leaking small amino acids should not be an issue.
Gary Lee Gilmore
Hi
I checked the composition of RPMI powder that I use. you are right! it was a good point to add sodium pyruvate. so I will do your recommendation.
Many thanks
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