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Transfection Forum Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


Any Transfection kit for HepG2 cells?

Any Transfection kit for HepG2 cells? - Transfection Forum

Any Transfection kit for HepG2 cells? - Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


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Old 08-20-2008, 04:06 PM
Pipette Filler
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Default Any Transfection kit for HepG2 cells?



Hi All
I have been trying to transfect HepG2 cells and I know they are diffifult to transfect. So far I have used methods for both transient and stable cell line selection without a great luck. The kits/methods I have tried are lipofectamine, lentiviral, nucleofection from Amexa, Retroviral and D293 DNA transfection kit from SignaGen. With all other other kits I have not have had any luck so far. I mean they work but the efficiency is terrible. I am trying to introduce siRNA as well as shRNA in to these cells. The only kit that impressed me was D293 from SignaGen. I got around 50-60% efficiency using GFP. But this kit is not for introducing siRNA as they told me there is some issues related to the release of siRNA from the transfection mix-siRNA complex. So they do not recommend this kit for siRNA transfection (that was disappointing). They have a kit for introducing siRNA but the best efficiency with that kit again is around 30-35%.

Does any of you have experience in using any transfection kit to transfect HepG2 cells, other than the above mentioned list successfully? Please get back to me if you have anything to share on how to get the best efficiency.

Any Help would be appreciated.
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Old 08-20-2008, 05:48 PM
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Default Re: Any Transfection kit for HepG2 cells?

Hello,
i have worked with Hepg2 cells for over 5 years. They are solid as a rock, and actually quite easy to transfect.

Fugene6 worked wonders, but also lipofectamine worked well.

How are you transfecting your cells exactly and what are you transfecting (size, amount)? Have you tried an siRNA DNA based construct?
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Old 08-20-2008, 06:34 PM
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Default Re: Any Transfection kit for HepG2 cells?

I have to agree with obWhat except to say that we get great transfection (of siRNA actually, leading to >90% knockdown) by doing a reverse transfection where where we seed teh cells with the transfection complex (there is a good protocol at Invitrogen). It is fast and easy and works really well for us.

Best of luck

Oh, we had good luck using both LF2K and RNAiMax

Last edited by amberpdidit; 08-20-2008 at 06:35 PM. Reason: forgot to mention transfection agents that work
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Old 08-21-2008, 06:04 PM
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Default Re: Any Transfection kit for HepG2 cells?

Thanks for your prompt reply. As I have discussed in my previous post, I have used lipofectamine too with not much luck. I must admit that I have not tried using Fugene. I am working on silencing transporters and reductases. My goal is to establish stable cell line as I have to do some arsenic based experiments on these cell lines. Using kits like lipofectamine would not be ideal for stables. I have tried shRNA DNA based construct. When I do selection with the antibiotic everything disappears indicating there was nothing inside the cells. Now you might ask how do you know the conc. of antibiotic was not toxic? I had done a killing curve and besides using the calculated conc. that was best for selection I have also lowered the concentration to see whether that changes anything. But no luck.
What is the transfection efficiency with fugene6? If you dont mind can you share your protocol for transfection?
As the transfection method for stable (shRNA:NA based) cell line did not work well, I had to revert back to siRNA and for that again I need somebody with a successful experience. Have you any experience working with HepG2 transfection reagent (Altogen)?
Again I an interested in stable cell line and for that I have all the shRNA's sitting in plasmids ready to go. I have wasted too much time trying to optimize nucleofection kit from Amaxa with only 8-10% efficiency even with GFP. Do you have any suggestions as to what other factors might be affecting this efficiency?
Right now I am attempting to use Retro viral based approach. I am not happy with even this approach. So any tip from your side would be valuable for me.
I appreciate your efforts.
Thanks a ton.

Quote:
Originally Posted by oBWhat View Post
Hello,
i have worked with Hepg2 cells for over 5 years. They are solid as a rock, and actually quite easy to transfect.

Fugene6 worked wonders, but also lipofectamine worked well.

How are you transfecting your cells exactly and what are you transfecting (size, amount)? Have you tried an siRNA DNA based construct?
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Old 08-21-2008, 06:07 PM
Pipette Filler
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Default Re: Any Transfection kit for HepG2 cells?

Have you tried transfection for stable cell line selection? To be honest when nothing else was working for stables I reverted back to siRNA approach. Thanks for you response. I appreciate that.

Quote:
Originally Posted by amberpdidit View Post
I have to agree with obWhat except to say that we get great transfection (of siRNA actually, leading to >90% knockdown) by doing a reverse transfection where where we seed teh cells with the transfection complex (there is a good protocol at Invitrogen). It is fast and easy and works really well for us.

Best of luck

Oh, we had good luck using both LF2K and RNAiMax
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Old 02-25-2009, 11:10 PM
Pipette Filler
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Default Re: Any Transfection kit for HepG2 cells?

You may try DNAFect 1.0 or LT from ATGCell. In our experiments, it significantly out-performed than Lipofactamine 2000 and Fugene 6 in IMCD cells.
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