I didn't use it in MCF-7 cells, but I used it in 293T cells. In my experience, Lipofectamine 3000 in transfection efficiency is the same as Lipofectamine 2000.

The transfection efficiency of 293T generally is quite high. But Lipofectamine 2000 didn't give good efficiency in MCF7, that's why I wanted to try lipofectamine 3000.

Dear Esther,

I would propose you an alternative to L2000 or L3000 that was demonstrated by several publications to work very well on MCF-7.

DreamFect and DreamFect Gold are two transfection reagents that are really efficient for many cell types, of whom MCF-7, you can refer to the following references:

Boguslavsky S., Proc Natl Acad Sci U S A. 2007 Jun 26;104(26):10882-7.

Caramusa L., J Cell Sci. 2007 Nov 1;120(Pt 21):3870-82.

Caramusa L., J Cell Sci. 2007 Nov 1;120(Pt 21):3870-82.

Ishikawa T., Thyroid. 2012 Aug;22(8):769-77.

Jiang H., Nucleic Acids Res. 2007;35(14):4767-78.

Smith NL., Cell Prolif. 2011 Apr;44(2):147-55.

do not hesitate to contact me if yo uwould like to try these reagents,

good luck with your experiments,

best regards,

Cedric

http://transfections.blogspot.fr/search?q=MCF7

Hi Esther,

We found that Lipofectamine 3000 has much higher DNA transfection efficiency than any other transfection reagents in MCF-7 cells, the transfection efficiency reached 60-80% (5Kb GFP expression plasmid) .

Here is the protocol (96-well plate format) :

The day before transfection:

Seed MCF-7 cells in 96-well plate at 2.4x10^4 cells/well;

The day transfection (Per DNA-Lipo P3000/Lipo 3000 Reagent complex):

0.1ug DNA + 0.2ul of Lipofectamine P3000 in 5ul of Opti-MEM (DNA: P3000 = 1ug : 2ul);

0.2ul of Lipofectamine Reagent in 5ul of Opti-MEM;

The total complex volume is 10ul --- 5ul of Opti-MEM(DNA-Lipo P3000) + 5ul of Opti-MEM (Lipo 3000 Reagent).

You can scale up and down the cell seeding numbers, the amount of DNA, reagent and complex volume for your transfection by comparing the growth area of your plate format with the 96-well plate (0.32cm^2/well). The growth area ratio will be your transfection scale up and down ratio.

I hope it will help you, please contact me if you have any questions.

Regards,

David

Hi David,

Thanks a lot for the answer. I will be trying out your protocol.

Best,

Esther

i wonder whether you would try to add or change the medium after the transfection as i found after one day cells dying...anyone occurs with this situation?

The new P3000 reagent seemed to precipitate my plasmid..  ~750 ul of Opti-MEM, 12.5 ug, 25 ul P3000..

Anyone else ever see this?

Yes even I observed that P3000 reagent seemed to precipitate my plasmid even with 3 ug

If you will add DNA in Opti-MEM before adding P3000 reagent then precipitation will not see.

The P 3000 must be added after the plasmid, if it is added before it causes precipitation