Try transfecting in media without antibiotics. They can enter the cell and cause toxicity during transfection with lipid-based reagents (lipofectamine etc). You could also try serum free (OptiMEM) medium. You can change the media after 4 to 16 hrs back to your normal medium. HEK 293 also typically become less adherent after transfection. I used to use the 293-AD line that adheres better. Good Luck!

It is quite normal to observe growth inhibition or even decreased cell viability post-transfection. Expression of your plasmid interferes with the proteostasis of your cells.

I've got a similar problem using Xtreme gene HP reducing proliferation in my cells.  I'm testing a few other transfection reagents and will let you know what I find. 

While you continue optimizing it, I would recommend lowering the amount of DNA (ug) you're adding.  For our cell line (PC CL3) we use 1ug DNA per well (of a 6-well plate) with a 1:2 ratio with Xtreme gene.  Sometimes we find that just 1ug is too much, so we co-transfect with an empty vector (but keep 1ug DNA total).

Other areas to trouble shoot are the cell density but I'm sure you know about that.

And just for reference:

http://www.roche-biochem.jp/sis/X-tremeGENE%20HP%20PackInsert.pdf

Let me know what you find, maybe it can help fix my problem too

Lipid-based transfections always caused some of my cells to die to varying degrees. I found using less reagent helped with viability

Try transfecting in media without antibiotics. They can enter the cell and cause toxicity during transfection with lipid-based reagents (lipofectamine etc). You could also try serum free (OptiMEM) medium. You can change the media after 4 to 16 hrs back to your normal medium. HEK 293 also typically become less adherent after transfection. I used to use the 293-AD line that adheres better. Good Luck!

Stephen's suggestions are quite appropriate. You can go for them.

But, it is just for the sake of my curiosity, I would like to ask, what is the gene that you are transfecting? Could anyway your gene might be interacting with the normal cellular growth and proliferation??

Thank you all for some great suggestions. First i will try Nylas suggestion,1ug plasmid, as i also felt that this could be an issue.

Thanks Stephen i avoid using antibiotics most of the time and during transfections especially, but a valuable point on how it can affect.

I am transfecting a transporter OATP1B1, it should not interfere with anything as far as i am aware, please let me know if you know otherwise.

Will be back if problems persist.

Cheers

Lower concentration of transfection agent could help. May be better to try different concentrations of Lipofect (or others) in OptiMEM without serum and antibiotics for 6h, then you can use your normal medium with serum, but still without antibiotics for 1-2 days.

Transient transfection will always have this problem. If you are doing transient transfection, then used inducible plasmid. Otherwise, i preferred stable transfection even though the level of protein expression is lower  

you could try lowering the DNA concentration even further than 1ug - we use between 0.1- 0.25ug plasmid DNA per 6 well and 1-2ul transfection reagent (Lipo 3000), we've also used a variety of others Lipo 2K (similar to 3k), Fugene HD (never worked well for us), Lonza nucleofection (very good-expensive). We mix lipid based ones in optimem but keep the cells in standard media (we tried keeping the cells in optimum but it reduced efficiency).

For Lipo, we've tested lipo alone effects on proliferation by EDU (FACS) and found it reduces proliferation (from no lipo) around 2-5% at 1ul, 10% at 2ul, 40% at 5ul and 50% at 10ul per ml. I'm sure this differs depending on your reagent but there will almost certainly be some reductions regardless.

For plasmids we've tested with GFP tagged proteins (2 separate membrane proteins) at ranges between 0.05ug-10ug, and found trafficking and expression were both better at concentrations below 0.25ug/ml. Expression is around 50%+ (by FACS). There are some significant drop-offs after that (to around 5-10%). We can get increased transfection at higher concentrations (1ug+) but confocal imaging shows the proteins junk up in the ER or incorporate to vacuoles. Overexpression of the proteins at these higher doses will no doubt stress the cells out and also probably lead to reduced proliferation.

Obviously these are just for our proteins but it may be worth checking in your cells. the main thing would be to make sure its trafficking correctly and not producing a toxic environment in your cells.

Hope this helps, 

Scott

Thank you Ali and Scott very valuable point, will take all your points into consideration.

I do agree with Stephan Fugs - try w/0 antibiotic,lipofectamine or Fugene 6 or HD are better transfection reagent for HEK 293 adherent cells with OptiMEM. For Fugene there is method for plasmid reagent ratio calculation for  transfection

Agree with the comments above that toxicity varies widely with the type of transfection reagent. Regarding adherence of the cells during and post-transfection, it may be helpful to plate your HEK293 cells on plastic coated with 0.01% poly-L-lysine. Best.

Great suggestions so far, so just a side note (and I'm not sure if this is relevant here, but); OATP1B1 is a transporter, in a class of transporters that can transport antibiotics INTO the cell. So if transfection using lipofection can 'co-transfect' the antibiotics, and disrupt cell growth, it's possible that expressing the transporter could do the same? You could do a mock transfection to compare (transfection) effect with (transfection + expression) effect on proliferation.

Cheers

I'd agree with many of the comments already posted. To add my tuppence worth, I would try reducing the concentration of your transfection reagent, as this can often have adverse effects on viability etc. We have had a lot of success transfecting HEKs with Attractene transfection reagent from Qiagen, this is again a liposomal based reagent. I'm sure they would be happy to provide a sample.

Xtreme gene HP is a very efficient transfection reagent and Hek 293 is a very easy cell line for transfection. I assume the transfection worked very well. Does your reagent control well also show the same effect (only reagent and no plasmid)? If not , probably you should consider the gene characteristic which u have inserted. The gene which u have inserted might have anti-proliferative effect (e.g. tumor suppressor genes ). If the reagent control well shows the slow growth, u can switch to Xtreme GENE( not HP).  Xtreme GENE is near to non-toxic. All the best.

Thanks for all your advice. Just let you all know, appear  managed to get things working. In the end all it required was a 700ug/ml G418 dose to kill all of the control cells and allow the resistant cells to flourish. All transfected cell line are now growing fine.

Cheers

I agree with Steven, culturing cells with antibiotics, especially during transfection, can negatively affect cell growth. A lot of it depends on what kind of plasmid you were using as well, and whether or not your transfection reagent was suitable for HEK-293 cells (like this one: https://altogen.com/product/hek-293-transfection-reagent-epithelial-kidney-cells/). Having optimal growth conditions still won't make the cells grow faster than before - transfection is stressful - but it can help prevent the loss of cell viability and slow growth.