Transfectiion of H295R cells by Fugene6 - (May/21/2006 )

Dear all,

I am currently facing the problems of transfecting H295R cells. I've tried different combinations of transfection parameters of using Fugene6. The best result that I've got from using cells of 60% confluency, and transfected for 48 hours. The efficicency was about or less than 10%.

May you guys offer me some suggestions or recommendations. I'll be much appreciate if you could give me a help!

Thank you!

Justin, a needy research student

Lipofectamine 2000 is very good, but as goos.. is expensive
did you try different ratios fugene:DNA? for me 3:1 was working fine for 293 cells using exactly the protocol provided in the kit.
But I stil lprefer CaPO3 as my cells are easy to transfect and is by far cheaper

I have worked with Fugene 6 but got nearly 75% transfection but then it could be bcoz of dif. cell line.

I agree with tertu abt lipofectamine 2000. I am using it for my transfections. Its expensive.

Well, Fugene 6 is not cheap either. What kind cell H295R is? I am working on developing transfection reagents. I will be happy to give it a try on some of the prototypes we have on hand if you like. I can get results in about 1 week. If you wish, you can send me a flask. These reagents are significantly better than Lipofectamine 2000 in efficiency and they are less toxic. Are you in US?

genehunter-1,

Just out of curiosity , what sort of reagents are u using for transfection, calcium phosphate or something else?

we are working on lipid- and polymer- based transfection agents. They have quite deversed structures. so far we have found several work very well on established cells and primary cells like HUVEC. I would love to test more cell types. Anyone interested to have their cells tested can contact me. As return, you will have the results, and free reagents from me. :-;

Hi,

I'm having exactly the same problems, have tried all the different ratio's also. We have had slightly better transfection efficiency using transfast, but still not that great. Can I ask which strain of H295R cells you are using, and if you are having any probs with culturing?

Have you also checked out Bill Raineys papers, he has had sucess using low serum media for 6 hours after transfection. I've still to try this, but the strain I'm using are quite slow growing.

Donna

I found this old post almost two years ago on this cell type. I am curious if anyone took his advice and tried GeneCarrier 1 and 2.

H295R Cell transfections -
(Sep/27/2004 )
Hi there,

I have been trying to transfect H295R cells which are human adrenal cells but am seeing no or very low levels of transfection even when using the pGL3 control plasmid which has a viral promoter. I hae tried FuGENE and TRANSFAST transfection reagents which are both liposome-mediated methods and have been reported to work in the literature. Does anyone have any experience of these cells or any ideas about what I should try?

Thanks very much

maza

-maza-
Hi,
I don't know anything about the cells you are using but with my cells which were difficult to transfect Lipofectamine helped a lot (from Invotrogen). You can also try the home-made Dextran sulfate method. It is ok as far as transfection goes, but is very toxic, so might be suboptimal.

-kant0008-
We have been using Epoch Biolabs' GenCarrier-1 and -2 (www.epochbiolabs.com) for various cell types, primary or established, dividing or post-mitotic, adherent or non-adherent.....They work very well for us better than lipofectamine2000, Fugene6 or GenPorter-2, and also cheaper!!
DEAE-Dextran only work in COS, monocyte and lymphocyte...

-postdoc2130-

My lab and the others have been using GeneCarrier-1 and -2 in our experiments and got very consistent results - high efficiency and low toxicity.

My colleagues next door, in their recent publication [Diabetes (2006) 55: 260-264], they have tried trasient transfection of human embryonic kidney 293T, HeLa, PANC1, and H1299 cells using genecarrier-1 and fugene-6 side-by-side, and their feedback is that genecarrier-1 is actually better and much cheaper. We used genecarrier-2 mostly on primary human pancreatic epithelial cells, it works very well. We have no experience on H295R cells. You may need to try it. Good luck.