I am transfecting E9 cells with K44a with pcDNA 3.1 backbone. The transfection reagent is PEI, the purity of the plasmid is 1.9 and conc is 340 ng/ul. I used 0.5 and 1 ug conc of Plasmid and 1.5 and 3 ug of PEI made up in plain DMEM per wells in 96 well plate and simulatenously did the same set up with PEI alone.
After 8 hrs of transfection, in wells transfected with plasmid + PEI, I could not find any cell attached to the surface and they are not even floating, but they are normal in PEI alone treated wells. They didn't recover even after supplementing normal media after 8 hrs of transfection. After 36 hrs the situation was worse, and all the cells had shrunk.
I used HiPurA endotoxin free plasmid purification kit.
This is common after transfection. To my experience, this is in part linked to the nature and the quality of the plasmid DNA. Sometimes, making a new prep of plasmid DNA may help. Reducing twice the amount of PEI & DNA per well also reduce toxicity. Finally you may change the medium of the culture around 5 hours post transfection. Good luck.
Have you tested to transfect the cells with a control plasmid? (e.g. one with a fluorescent protein). Then you would know if the problem is with the DNA, gene or transfection reagent. We do not normally have problems with PEI at least we use very high quantities.
Did you transfect with vector as control? I have cells that handle the transfection reagent but not plasmid yet they electroporate with high efficiency. Have you transfected the cells before? Another option is that K44a may be toxic to the cells for what ever reason. I have also experienced this that one cell line dies when transfecting yet another survives with the same gene - have you tried a second cell line?
E9 cells are very sensitive to endotoxin. Extract plasmids with QIAGEN® Plasmid Maxi Kit [Cat. No.12162; Lot. No. 42792199] and try once more. Good luck!
When you do transfections, an untransfected control, empty pcDNA vector control and a only transfection reagent control is required to know where the problem lies. Toxicity is a common problem with synthetic reagents. From the confluency of the cells, to the condition of the cells during transfection everything will matter. what we follow is a 4-6 hour incubation time for cells in a transfection medium (DMEM+ 1% L-glutamine and not only DMEM). TF medium again depends on the kind of transfection reagent you use and even the incubation time will vary there.
You can see the references as to what kind of transfection reagents are used. CaCl2 method of transfection is conventional but non toxic. We use lipofectamine where again toxicity is there but ration of plasmic to lipo has to standardized based on kind of experiment.
So before fixing a ration, simply GFP plasmid can be transfected from an increasing ration of DNA:TF reagent and fluorescence can be checked where there is maximum expresssion and minimum cell death or rounding up. That ration can be used in the same cell line but with your plasmid DNA construct.
We also use Endotoxin free kit from Qiagen. Its good. So try using this and donot forget to have your vector control, and manage the ration of plasmid to reagent.
Hope it helps.
Cheers
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