I think you are using too much Lipofectamine for transfection. Try transfection in 10% DMEM or RPMI medium, no need to use serum free medium. Dilute your DNA and Lipofectamine mix in Serum free medium. Protocol generally says using 1-2ug DNA with 6-8ul/well lipofectamine in 6 well plates. And change the medium after 24 hours of transfection. Dont forget to use high quality DNA for transfection (Maxi-prep DNA)

Use any GFP plasmid DNA to optimize the condition to see transfection efficiency and toxicity. 

Have you tried removing the media that contains the transfection complex 6-24 hours post transfection and replace it with growing culture media? If this doesn’t help, maybe try a small experiment to optimize your transfection. Cell density can be an important factor. If cell density is too low at the time of transfection, then a big drop in viability may be seen. Also, as others have emphasized, ensure that high quality DNA is used. I would also test two different cell densities, the two recommended reagent doses and two DNA doses - if possible.

Have you tested this DNA on any other cells with success?  Some other things to consider are the cells themselves...are the cells healthy?  It's also best to transfect the day after seeding.  

There are two components in the Lipofectamine 3000 kit – Lipofectamine 3000 and P3000 reagent. When you perform DNA transfection using  Lipofectamine 3000, diluted DNA need mix with P3000 before combining with diluted Lipofectamine 3000 reagent to make complex. Without P3000 reagent in the complex, you will get very low or none transfections on cells.

For MCF-7 cells, we always get ~55% transfection efficiency (GFP plasmid). Here are some suggestions for delivery DNA to MCF-7 cells by using Lipofectamine 3000 (24-well plate format):

The day before transfection, seed 1.5x105 MCF-7 cells per 24-well plate well , 85% confluence at the time transfection

Next Morning, transfect the cells ( for each transfection reaction):

Changing cell media at post-transfection 6hrs is not required for using Lipofectamine 3000, but if the cells are sensitive, such as MCF-7, to transfection, medium changing will help to reduce the cell death.

ATCC MCF-7 cells growth rate is low. We use Gibco DMEM, high glucose, GlutaMax Supplement, pyruvate + 10%FBS to culture the cells. The supplement sodium pyruvate will help MCF-7 growth a lot.

Hope this will help you.

Best,

David

Hi David Wang, Sir I do not have opti-MEM media as of now. In that case, I will dilute the Lipo3000 and DNA-P3000 in serum free MEM medium. 

My concern is which media should I use to incubate MCF7 cells for 6 hours incubation with DNA-Lipo comples...should it be serum free medium or reduced serum medium?

As after 6 hours, I will be replacing it with complete growth medium. Please suggest.

Yes, you can dilute Lipo3000 and DNA-P3000 in serum free MEM or DMEM medium to make complex, but it will have less transfection efficiency than Opti-MEM I.

The media for MCF-7 cells during transfection is the complete MCF-7 growth media which has 10%FBS, such as DMEM/GlutaMax/pyruvate with 10% FBS.

Post-tfx 6hrs, change the cell media by removing media containing transfection complex and replacing with fresh MCF-7 complete growth media.

Please let me know, if you have more questions,

Best,

David

Hi everyone, I have a question: Does p/s affect transfection efficiency? I usually add p/s to DMEM (along with l-glu) and use it to transfect HEK293 cells using lipofectamine 3000 but I haven't been able to see any protein expression when analyzing the medium. However, I am afraid that not using p/s will cause bacteria to develop. Any suggestions? 

Does it also work when I add the DNA to the LF3000 dilution and mix with the p3000 dilution in a second step?  Or does the DNA need to see p3000 before it is put together with LF3000?

Hi Dear David Wang ,

How much DNA and lipofectamine I need for 175 cm2 plates? 

Thanks

Hello Everyone

I have question , I used six well plate for the transfection of my plasmid, according to the lipofectamine 3000 Kit, the total volume should be 250 μl of DNA-Lipid complex, is this volume enough to cover the cells in each well for six hours transfection or I should add more medium to the cells before the transfection?

Hello Heba

I think you meant that you add only 250 μl of DNA-Lipid complex to each well.

No, each well in the 6-well plate should contain 2 ml of the standard growth media plus the 250 μl of DNA-Lipid complex OR (1750 μl of the standard growth media + 250 μl of DNA-Lipid complex).

Dear Prof kamal try to decrease the incubation time.

Hello Kamal

Thank you, Yes I did that but the maximum transfection efficiency which I got is 30% only, is it normal?

Hello Heba

Try to increase the amount of DNA plasmid in each well OR increase the incubation time.

Thank you very much and can you please tell me if you have experience in the cancer biology field which cancer cell line over express EGFR oncogene?

Hi

What is this P3000 reagent and How it helps in Transfection, is it something which drives DNA to Nucleus.

Any one have idea about How P3000 Reagent works

Ashwni Verma,

As I understand it, p3000 is a lipid which coats the negatively charged DNA. Lipofectamine 3000, when added to the mixture coats p3000 lipid droplets and confers a positive charge to the outside of the liposhperes. This positive charge allows endocytosis of the lipid droplets into the negatively-charged cell membrane.

David Schad, Doesn't this mean that the P3000 reagent and the lipofectamine 3000 reagent are redundant? what does the P3000 reagent do that lipofectamine does not, and vice versa?

I ask this because one of my supervisors told me that it is okay to transfect without the P3000 reagent (I am transfecting lenti into HEK293FT cells btw)

Andrew Kim,

That is a good question, and I don't know the answer. Indeed, most other transfection reagents are one-component. I am interested to know if omitting p3000 still gives you similar transfection efficiency.

Hi, Andrew Kim, I would like to add some.

Even though the P3000 reagent helps Nucleic acid to enter the nucleus, well per my experience, the P3000 reagent is not beneficial for many cell types as it does not enhance any significant transfection efficacy and in the case of lenti-transfection it will work without P3000 reagent coz lipofection is enough (for me it works in fibroblasts). I used HCF and HL-1 cells, I didn't use P3000 reagent for that, and transfection (plasmid) works very well. However, my colleague's transfection experiment does not work without the P3000 reagent (he used cardiomyocytes and the plasmid size is quite big almost 9.4KB).

Iqra Mushtaq that is interesting... so P3000 is just sort of like a performance-enhancer so to speak for bigger plasmids? at least from what I'm getting from you and your colleagues experience...