Bacterial contamination is problematic for cell viability, but it does not necessarily lower transfection efficiency. Given your results, it doesn't actually seem like you had such bad efficiency overall. Our HEK293T reagent, which we optimized for HEK293T (https://altogen.com/product/293t17-transfection-reagent-emb-kidney-crl-11268/), has 90% efficiency. If you're trying to go anywhere higher than that you'll likely just encounter cell death. If you need a more homogenous population then you should look at some selection protocols (puromycin resistance, etc.) and use those to get a population where all cells have been transfected.

How much lipofectamine are you using? try to reduce its volume if possible.

In case you are having antibiotics in your cell culture media, try to avoid it, it often improves the cell survival rate.

Cell death may also be due to the overexpression of your protein of interest...against this you cannot do much. In order to test whether your viability problem comes from the transfection reagent, try another one like the inexpensive calcium phosphate or fugene.

You may also consider the reverse transfection, it also gives good results.

Actually I don't see a problem in the transfection efficiency , 80-90% GFP after 24h looks quite good but it seems that you have a problem in your transfection procedures.

How much L3000 do you use per well?

Did you ensure that your transfection complex volume is enough to cover the whole well surface?

Did you change media 4-5 hours after transfection?

Hi Cyrille,

I am using 9 ul lipofectamine. Previously I used lifeact GFP with 6 ul lipofectamine and 3 ul plus reagent. I got 80% transfection efficiency but whenever I use this R8.2 plasmid I do not get good result.

Hi Mohamed,

I use 9 ul L3000 if I take 3 ug DNA.

If I take 1-2 ug DNA I use 6 ul L3000.

Total 250 ul transfection complex I add in 2 ml of DMEM + 2x10^5 cells/well.

I did not change the media. I got good result without changing the media but using lifeactGFP.

hi

I do the transfection with media DMEM without serum and I remove the media after 5 h on incubation 37ºC. then I use DMEM 10% serum. Some protein need growth factor for the expression. hope something help you.

Hi

Plate cells at 40% confluency in 6well plate and culture overnight. Wash double cell thoroughly with PBS to remove any remaining serum-derived proteins.

Transfect with your protocol by gentle, sufficient mixing them. After 6h medium change and then allow to grow or die for 48h.

Wash thoroughly and add drug for 10 days (change the drug every 48h). Highly overexpressed cell will die to the toxicity, only the affordable gen copies of cells will survive as stable transfectants.

if its a toxic protein in high expression Can you do three different concentrations of 1, 2.5, 5 and 10ug DNA/ml for transfection of the same cells

'' Dr,Sang Ho Lee''

It worked in my case

BEst

Hi Oscar ,

I tried the transfection with DMEM (without serum and antibiotics) but the efficiency did not improve.

I also tried the transfection with reduced amount of lipofectamine suggested by Cyrille and Mohamed but the efficiency was same in every case.

Any suggestions are welcome.

Thank you all. It seems my cell line had bacterial contamination.