How to keep cells healthy after transfection - Any tips? (Nov/27/2006 )

I've realized that my cells are not healthy during/after transfection, which I think is either contributing to low transfection efficiency or, more likely, low protein expression.

My protocol:
I use 293T cells and usually transfect in a 24 well plate, but would be willing to use a larger plate.
I use Fugene 6 at various Fugene:DNA ratios.
The cells are usually ~70% confluent at the start of transfection, and they are not becoming over confluent after 48 hr culture. In fact they are not growing much.
I have been splitting the cells in serum(+) media and then changing to serum(-) twice on the day of transfection, so the transfection is done in serum(-), antibiotic(-) media (DMEM:F12). Then I want to culture 2 more days in serum(-) media.
However, the media is turning yellow after 1-2 days and cells don't look good. The media is not contaminated (that I can see) and cells are mycoplasma free.
I can transfect in serum(+) media, but I must culture at least 2 days in serum(-) before collecting the media.

Questions:
What is the optimal media for transfecting/culturing 293T cells? Is there any media that the pH would stay okay over the 2 day culture time?
Are there supplements I can add (other than FBS or BSA) that would help my cells stay healthy?
If I decide to change media after the transfection to get rid of Fugene and add back antibiotics, how long post-transfection should I do that?

Thank you so much for any help and advice, I really appreciate every comment.

You can (easily) transfect 293-cells in media containing serum (I've known some to do it in media containing pen/strep, so adding back antibiotics soon after transfection will not alter your results I believe).

Apart from this, you might wanna try growing them in serum free media, they will need to adapt before you can do transfection of course. adaptation takes some time. I know GIBCO has some serum free media, probably other supply it as well.

i do mine like this
seed my cells in antibiotic free media but serum+ then next day do transfection and after 6 hours change the media into my normal serum+ antibiotic + media for 2 days
the only media i use that serum- antibiotic- is the one i use for preparation of my plasmid anf lipofectamine.
hope this help.

I used to transfect 293 cells at a way lower density. I used 40 % - 50 % density at the day of transfection. But it might depend on when you want to examine the cells, I usually examined them 48hrs after transfection.

Thank you, everyone for your replies. I'm going to try some lower confluencys as suggested and do the transfection in serum(+) media and change to serum(-) after transfection. I'm also slowly adapting some cells to lower serum conditions and eventually to serum(-) as suggested so hopefully that will improve the health of the cells after transfection.

The problem is that I must culture the cells in serum free media for at least 1-2 days post-transfection becasue some serum proteins are the same size as my protein of interest and interfere with my western blot.