how to avoid MEF when mES cells are transfected by lentivirus? - (Jun/05/2006 )

Hi,there, I will tranfect the mES cells with lentivirus, as you know, lentivirus can tranfect non-dividing cells as MEF, so I wonder if I should culture the cells in a feeder-free system to avoid this problem? but mES cells cultured without feeder will differentiate easily, does anybody have some suggestions?

Thanks!

Jerrylee, I routinely culture mES cells without feeders. If you have a good batch of LIF you dont really need the feeders. Just remember not to plate the ES cells too thinly as this is a sure way to make them differentiate.

Hi,Techqueen,Thank you for your advice,I'll try.Do you mean to plate the cells at a high density?We use a batch of LIF from ERSGO at 1000u/ml,I wonder if you could let me use your protocol?
fmli@sibs.ac.cn

[quote name='JerryLee' date='Jun 8 2006, 03:39 AM' post='54608']
Hi,Techqueen,Thank you for your advice,I'll try.Do you mean to plate the cells at a high density?We use a batch of LIF from ERSGO at 1000u/ml,I wonder if you could let me use your protocol?
fmli@sibs.ac.cn



I have never used this type of LIF but it is pretty popular.

As far as passaging your cells I usually split at about 70-80% confluence at no more than 1 to 5, but there is a bit of leeway here.

What else is in your medium ? I take it that it is bog standard ES Cell stuff. If thats the case you should have no problems.

Hi,Techqueen, the stuff in my feeder-free medium are as follows:
DMEM high glucose (GIBCO);
0.1 mM non-essential amino acids (GIBCO);
1 mM sodium pyruvate (GIBCO);
100 microM beta-mercaptoethanol (Sigma);
15% fetal bovine serum (Hyclone);
Penicillin and streptomycin 50 microgram/ml each （GIBCO);
L-glutamine 2mM (Gibco);
1000 U LIF/ml (Chemicon);




[quote name='Techqueen' date='Jun 8 2006, 08:49 PM' post='54682']
[quote name='JerryLee' date='Jun 8 2006, 03:39 AM' post='54608']
Hi,Techqueen,Thank you for your advice,I'll try.Do you mean to plate the cells at a high density?We use a batch of LIF from ERSGO at 1000u/ml,I wonder if you could let me use your protocol?
fmli@sibs.ac.cn



I have never used this type of LIF but it is pretty popular.

As far as passaging your cells I usually split at about 70-80% confluence at no more than 1 to 5, but there is a bit of leeway here.

What else is in your medium ? I take it that it is bog standard ES Cell stuff. If thats the case you should have no problems.
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