I transfected HCt116 cells with GFP using Lipo3000. I didn't change media after transfection as the manufacturer guidelines say not necessary. However, I noticed a change in the cell morphology and failed to visualize any fluorescence from GFP. So I was wondering whether the Lipo became toxic to the cells because I didn't change the media after transfection and left it for overnight incubation. Could that be the reason? Has anybody else faced this problem in this particular cell type? I would greatly appreciate your thoughts or valuable comments on this.
Thanks in advance
Hi Gowthami Mahendran
Briefly, if your cells are too sensitive one, L3K is gonna be toxic. Consequently, changing the medium after 6 to 24 hours would be helpful.
Other factors that take a role in the observation you mentioned:
1. The plasmid itself, some plasmids are more toxic than others. I have not found an article supports this but it is from my experience.
2. The PH of your medium affects the GFP intensity. In other words, you may have positive cells with a break-down GFP, consequently you consider them as negative cells by optical observation.
If you are worried about the efficiency, actually there are a bunch of optimization you can go with:
1. The ratio of plasmid/L3K.
2. The concentration of FBS. Each cell line has its own capacity to be transfected with different FBS%.
3. Try different time points to observe the cells. In our case 24 to 48 hours was optimum periods for the highest protein levels.
Another suggestion that may be helpful (also without any supportive article), one-strand plasmid has higher transfection efficiency and protein level than double-strand one.
In addition, the location of the protein of interest is important, cytoplasmic protein are expressed in higher amount than the nuclear ones.
Hello Gowthami Mahendran
Usually complete media change may be performed 5-24 hours after transfection. The manufacturer guidelines is right. But you should also note that certain cells are sensitive to lipofectamine 3000. In such a case, if HCt116 cells are sensitive to lipo 3000 it becomes necessary to change the medium 4-6 hours post transfection as it will help reduce cell death. Try to change the media 6 hours post transfection.
Best.
Thank you Mohamed Khashan and Malcolm Nobre for the great suggestions and elaborate explanation. I'll try them out and see
Not necessary to chage the media after transfection. But If cells looking more prominent towards death then you have to change the media.
Hi Mahendran,
I have experience in transfection using lipofectamine 3000,LTX and 2000. There are some factors responsible for successful tranafection. The first one is your type of cell lines which some cell lines are resistant toward lipofectamine, and the division rate of cells are another factor which if they be in lower passage you will have more chance to successful transfection. Regrading your question, it would be better to change your medium 3 hours before transfection to put your cells in a starvation state. If you have further questions you can contact me directly.
One more point that, you should change your medium after 6 hours of tranafection because there will
be no further diffusion lipofectamine/DNA complex into cells, and many cell lines are sensitive to lipofectamine which may be toxic for them.
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