U2OS transient transfection - (Dec/01/2009 )

Hi,

I've been trying to transiently transfect U2OS cells to carry out co-IPs using lipofectin/lipofectamine reagents. I've worked with 293T cells and I managed to always get good levels of transfection, without having to worry too much about cell density or reagent:DNA ratio, but with the U2OS I can't seem to be able to establish a procedure that guarantees me good transfection rates every time. I've tried playing around with everything I could remember but I'm still not getting it to work. Does anyone have experience with this particular cell line?


Thanks!

I also got the impression that U2OS are a little touchy in terms of viability after transfection and efficiency - I swicthed to siLENTFECT some time ago which gave me much better efficiency than lipofectamine. Maybe you want to give that a try (you can use it for anything, not just siRNAs).

warsel on Dec 1 2009, 10:32 AM said:

I use lipofectamine 2000 for U2OS and usually get around 50-70% efficiency in U2OS for plasmids and 80-90% efficiency of siRNA. Co-transfection of plasmids and siRNA results in reduced efficiency of plasmid delivery, but similar siRNA delivery.
I've also used silentfect for siRNA in U2OS with >90% efficiency.

@warsel, you say that siLENTFECT works for plasmids as well? What's the efficiency like for plasmids? Why doesn't BioRad advertise this? Have you used it for co-transfection?

I am sorry, I missed your post here.

Yes, siLentfect works just fine for plasmids.
I am also wondering why BioRad does not advertise this.. but that may have some marketing reasons - I really don't know.
We do all our plasmid transfections with silentfect since that seems to work better than all other lipofection reagents (at least for the cells we are using).

Dr Teeth on Dec 2 2009, 09:06 AM said:

@ Dr Teeth or others
What conditions did you use for transfecting U20S with lipofectamine ? concentration of plasmids and number of cells ?

Hi,

I have been working with U2-OS cells since some months now and also had problems getting a good efficiency of transfection. Some literature says that they have a regulation of PH that affects the expression of the protein. However, with siRNA the case is different and they are comfortable. I used FuGene6, HD, Lipofectamine and found that lipofectamine and FuGene HD yielded about 60 % efficiency and then I switched to Electroporation device from AMAXA-Lonza and I was able to push it up to 85%. However there is a considerable cell death and hence one must pay attention to the number of cells he needs in the end (approx 15% loss). But its not much different with Lipofectamine! My cells also died upon high Lipofection reagent concentrations (high reagent to DNA ratios). Alternatively one could also consider using Opti-MEM media for transfection. It was better than the Mccoy media that was recommended.

Hope this helps!

Cheers!
Prem

I do lot of SiRNA and mRNA transfection. For better results use metafectaneSI kit from Biontex Inc. Try out the free test samples. It works for you