6 replies to this topic

[aalvesdu]

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!

[warsel]

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).

[Dr Teeth]

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

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).

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?

[warsel]

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 [mostly 96-well lentivirus packaging] with silentfect since that seems to work better than all other lipofection reagents (at least for the cells we are using).

[haveaquestion]

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

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

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).

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?

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

[Biobaba]

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

[krenny7]

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