Hello everyone
I have never cultured cells in my life and everything is new to me. I've done some tests with Lipofectamine® 2000 in my IMR-32 cell line with some plasmids containing dTomato that we designed (they carry the promoter and enhancer we'd like to study). I followed the protocol instructions and let them mix (DNA + lipofectamine) for 30min in serum-free medium. So far my maxium transfection efficacy according to cytometry has been 0.94%!!
What can I do to improve such low number?
For 6-well plates I've been doing:
150 uL Opti-MEM + 6/9/12/15 uL Lipofectamine (12 was the best)
150 uL Opti-MEM + 14 ug DNA
I tried 30 ug DNA and the results were even worse, so I don't know...
Thank you very much in advance
Bea
i would like to just give a hint that you might be using too much DNA. In a 6 well plate i think the protocols of Lipofectamine recommend 2.5 ug DNA
My go-to/starting recipe for a 6-well plate is 2 µg of plasmid in 100 µl Opti-MEM and 6 µl of Lipofectamine 2000 in another 100 µl Opti-MEM per well. Mix, incubate for 5 minutes, and add dropwise to the well.
The optimal ratio can vary by plasmid size and cell type. In addition, some cells are just not amenable to transfection (e.g. H2009 lung adenocarcinoma cells are poorly transfectable while H1299 lung adenocarcinoma cells lines transfect easily)
I'd also recommend measuring your transfection efficiency using a strong constitutive promoter like CMV. It might be that the promoter you are investigating is only weakly active in your cell line.
Hi everyone
Thank you both of you for your very kind answer!!
You were right, it was too much DNA. I was using 14 ug cause that's what the protocol said, but I tried again with fewer DNA and I improved from 0.94% to 3.28% with 2 ug DNA :) !!
It is of course still very low but my cell line won't probably get waaay better with my plasmid since I tried GFP-CMV-bActin and I got 18.69% maximum...so it's not like these cells get to 70% easily, it seems. At least with Lipofectamine 2000
Thank you again!!!!! If you have any more advice to still improve this 3.28% I'll still be thankful.
Bea
If you're not already performing your transfections overnight, you can give that a try. In cells that transfect well, only a few hours will give high efficiency, but in some cells a longer incubation is necessary.
You can also try performing your transfection in serum-free media (e.g. Opti-MEM) if you cells can handle it. Serum contains a lot of charged proteins that will bind to the lipid nanoparticles and may reduced their uptake.
In addtion, you can try other transfection reagents. Lipofectamine 2000 is a fairly robust system, but sometimes others will work better. I don''t have any other systems I would recommend with any supporting evidence, but I've also used Lipofectamine 3000 and X-tremeGENE HP Transfection Reagent with good success in various cell lines (and primary cells). These reagents are not cheap, but you may be able to get a sample from your suppliers just to validate on your cell line.
Finally, you can try some other methods of gene insertion. Electroporation is highly efficient but also very harsh on cells. Adenoviral vectors are also very good for transient expression, but they require quite a lot of work to generate compared to simple plasmid transfections.
I check the cells 48 hours post transfection so they spend quite a time with the Lipofectamine and DNA... it doesn't seem to be toxic for them so I just leave them alone for 2 days. They are of course in DMEM GlutaMax with FBS and antibiotics, not in Opti-MEM, I could try that I guess...
If you do a transfection using only Opti-MEM I would recommend changing back to the complete culture media the next day.
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