HeLa transfection - using Lipofectamine 2000 (Nov/08/2005 )

The thing with transfection reagents is: they are toxic to your cells. You have to get something (DNA, RNA, ...) through a cell membrane, which isn't supposed to let these substances pass through. Also, DNA in the extracellular environment off cells is toxic too (in you body, DNA should be contained within the nucleus of living cells, or just within the cells when they are dividing so DNA outside cells is a signal of necrosis or infection).

So removing DNA and a substance that is (to some degree) toxic after a couple of hours is better for your cells. Your exposure time is reduced this way. Suppliers say that the stuff isn't too toxic, but when you see comparative studies by companies, there own product is almost 0% toxic whereas the ones of other suppliers are always very toxic (a lot more than they claim themselves).

Some other stuff you should be aware of: how was your plasmid prepared? (assuming you are transfecting plasmid DNA). Most plasmid preparations give you good quality of DNA for restriction, pcr and other basic molecular biology handlings, but for transfection you better try out "endofree" DNA. Check out www.qiagen.com for more info on endotoxin free plasmid preparations (there are probably other suppliers of endotoxin free DNA preparations as well).

Apart from this, try doing the following negative controls: transfect without DNA, and transfect without lipofectamine. This way you can to some extent find out how toxic one or the other is.

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Hi,

Thank you very much for thi elucidative explanation.
Once again you are absolutely right, DNA itself can be toxic!
And I should check the quality of my midi-prep. Nobody ever mentioned me about the endotoxin problem. I always do plasmid isolation using a kit from Macherey Nagel (Nucleobond columns).
I will check up the QIAGEN web page.

Thank you very much,

P.S amazing you work for so long with something and still there are basic things that you don't know. Good I found this bioforum, along with google and PubMed is the best site I know!!!

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Hi

Today, 24 hours after transfection. HeLa cells are all dying. I have tested different concentrations of DNA and lipofectamine.
Changed medium before and after transfection, no difference.
Maybe I should use Opti-MEM, that's what I will try next.

Meenwhile if anyone remember any other sugestion please let me know.
Maybe I should also thaw a new batch of cells.

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If you check out
invitrogen's protocol you find last tips for transfectioni of Hela cells using lipofectamine2000. I'm gonna try this way next week (but as I'm going for homologuous recombination of a virus in these cells, it takes some time to see if it's efficient).

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Hello again,

I went to the Invitrogen website and in fact they recommend the use of Opti-MEM to diltute DNA and Lipofectamine 2000. That is what I will do next time.

One thing I saw for tje first time.
Invitrogen says that the highest transfection efficiency of HeLa cells is obtained with Lifectamine Plus.
For a long time ago I have been asking my supervisor to change to Lipofectamine Plus ( I used in another lab and it worked so much better!) but she didn´t want because was more expensive.
I will try one more time transfection of HeLa using Opti-MEM if I keep having so many cells dying (50 % or so) I have now reason to ask again to change to Lipofectamine Plus.

Thanks

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Okay,

as said, I transfected according to Invitrogen's protocol (serum-free transfection). I did however replace my medium (with a PBS wash step) after 4 hrs.

In one plate I used a plasmid (about 14 kb) that wasn't purified Endofree. In this plate I noticed that 2 days after transfection most of my cells were floating around, but they were all green (the plasmid contains an EGFP, so...).

The other plates, I used plasmids that were endofree + PCR product, and here there were hardly any cells in the medium.
As this was a transfection for homologuous recombination I need to grow the cells a lot longer to see sufficient expression of a marker gene, I'll keep u updated.

So, for me, this indicates that the endotoxins are a real issue, and that transfection with lipofecamine2000 is very efficient when u folloow invitrogen's protocol.

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Me once again. As said, I did transfection for homologuous recombination, and now (monday morning), about 5 days post-transfection, I'm seeing expression of my marker gene (I do HxR of plasmid and a PCR product, the plasmid contains EGFP). This is something I haven't seen before.

So, the difference between this time and the other times was that I diluted my DNA and Lipofecamine2000 in Opti-mem instead of RPMI, that I plated 2,4 million instead of 2 million cells day before transfection, and that I used serum free medium during transfection instead of medium with serum. It seems my overall succesratio has increased significantly!

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Hi,

This forum is full of really good and useful informations ! I've learned a lot about how to improve my own Lipofectamine 2000-mediated transfection. I am actually using different cells than Hela, namely rat fibroblasts and have a lot of toxicity too.

After trying different amount of Lipofectamine reagent as well as DNA, it seems obvious to me too that DNA and Lipofectamine are really toxic. Still I think this is not all of my problems. I definitely want to test changing the medium 4 hours after transfection and I may also try adding serum, something like 5%, during the transfection step itself... We'll see.

I have actually a question related to the plasmid used to transfect the cells. I read somewhere that it was better to cut the plasmid once before transfecting it when you'd like to perform stably transfections.
Does anybody know, from experience, if it is true? Is the efficiency of the transfection better when the plasmid is circular or cut? (I extracted my plasmids using one of the endofree kits from Qiagen but as I cut them, I purified them ultimately only with a regular DNA purification kit, which might also explain my high toxicity...)


Thanks for your suggestions !


M-kun

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Hi

I would like to thank you all and particularly Vairus for all the tips.

Now my transfection efficiency is higher and more importantly cells are healthier as the % of surviving cells is much higher.

The difference are:
- I dilute DNa and lipofectamine 2000 in Opti-MEM.
- just before addind DNA-Lipofectamine complexes to cells, I change culture medium (DMEM+10% FBS) by OptiMEM.
- 4 hours after transfection, remove OptiMEM, rinse cells in PBS and add fresh culture medium.

For 96 well plate for me worked well plating approximately 1000 cells /well
transfecting them with 300ng DNA and 0.5 ul lipofectamine 2000
(=DNA-Lipofectamine ratio of 1:1.6)

Thank you and good luck for all of you with your transfections.

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Hi, i have looked through all your messages!

I am doing transfection using Lipofectamine 2000 and plasmid DNA, too. I do have cell toxicity, but not so serious as yours.

What I found is Lipofectamine 2000 is toxic to cells, so I reduced the recommended quantity of the Invitrogen protocol.

You can not use antibiotics during transfection step because it will cause cell death.

The same as yours, I haven't try to use Endofree kit to prepare my DNA, maybe that is the problem!

Even I had some survival cells, my knocking down experiments didn't work so well. I tried several time, each time I got different results!

I am feeling frustrated now!
Hope we can discuss and find some helpful solution!

Berry

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