Cells are dying after transfection due to toxicity of either DNA/ lipofectamine reagent
What specific media should be used while diluting the lipofectamine reagent(recommended opti MEM or any reduced serum medium)
And, how does it affects the efficiency?
Hi Amit, I have used Opti-MEM from Invitrogen for transfecting HEK-293 cells and it worked well. Few other things that I tried optimizing were: ratio of DNA and the lipofectamine reagent as suggested in their instructions. I was getting very low transfection efficiency - what helped was seeding cells at a low confluency (~50-60%) and using high concentration of plasmid DNA. About 80-90% cells were successfully transfected.
I think you are using too much Lipofectamine for transfection. Try transfection in 10% DMEM or RPMI medium, no need to use serum free medium. Dilute your DNA and Lipofectamine mix in Serum free medium. Protocol generally says using 1-2ug DNA with 6-8ul/well lipofectamine in 6 well plates. And change the medium after 24 hours of transfection. Dont forget to use high quality DNA for transfection (Maxi-prep DNA)
Use any GFP plasmid DNA to optimize the condition to see transfection efficiency and toxicity.
OptiMEM is the best media to use for transfection, but this is just used for complex formation between the reagent and the DNA. The cells should still be in complete media, as previously mentioned, when the transfection is added to the cells. The media used for complexation can affect the efficiency of transfection, but it's usually pretty minimal (5-10%). The one factor I would consider for your complex media, if using OptiMEM, is the color of the OptiMEM. If it is a purple color, this can have a negative effect on efficiency, but I wouldn't say this to be the reason for the toxicity that you are seeing.
What cells are you trying to transfect? Is your DNA clean and endotoxin free? have you tested it before in an easy to transfect cell line? Have you tried changing media 6-24 hours post transfection? If this doesn’t help, maybe try a small experiment to optimize. Cell density can be important because if too low at the time of transfection, then a big drop in viability may be seen. Also, as others have emphasized, ensure that high quality DNA is used. I would also test two different cell densities, the two recommended reagent doses and two DNA doses - if possible.
Hi everyone, I have a question: Does p/s affect transfection efficiency? I usually add p/s to DMEM (along with l-glu) and use it to transfect HEK293 cells using lipofectamine 3000 but I haven't been able to see any protein expression when analyzing the medium. However, I am afraid that not using p/s will cause bacteria to develop. Any suggestions?
Vanessa, my interpretation is that the increased membrane permeability induced by lipofectamine can allow some antibiotics to pass into the cells and induce cytotoxicity. Antibiotics should, therefore, be avoided when transfecting. With good aseptic technique, there should be no need for them, certainly for this length of time.
Amit, this may help:
https://www.thermofisher.com/uk/en/home/references/gibco-cell-culture-basics/transfection-basics/factors-influencing-transfection-efficiency.html
Use extreme gene HP. I get more than 80-90% transfection efficiency in HEK293T. You do not even need to change the media after transfection.
I use 2.5ul of Lipofectamine 3000 for each 1ug of plasmid each in 50ul optiMEM incubate for 20 min at room temp, then add to 1^6 of HEK293cells that plated in DMEM supplemented with 10% FCS, 48 hours later collect media. I got 90% efficiency.
Good luck
Hello Nikita
My protocol is for 24-well plate, using HEK 293 cells and P3000
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