FuGene 6 transfection problems - (Mar/06/2008 )
As anyone ran into problems using FuGene 6 for transient transfections in adherent CHO cells?
I've tried using several DNA: Fugene ratios to determine the optimal amounts however, when I attempt to verify my transfection efficiency, I yield something close to 0.
Here's the protocol that I'm using:
1. Make the DNA:Liposome complex in serum-free media
2. Incubate for approx. 45 minutes at RT.
3. Add transfection mixture to the plate.
4. Return plate to 37C
5. Change media the next day
any thoughts on what I'm doing wrong or what I can possibly try??? I'm running out of options. 
Sometimes if DNA quality is not good or pure, then transfection doesn't work properly.
Always start with a simple reporter, like EGFP, pCMV luciferase or beta-Gal that you know works.
Is that incubation time too long?
This is the protocol that I use when doing transfections with FuGene 6...
1. Combine the FuGene and SFM in one tube. Quick vortex, tap tube to settle, and sit 5 minutes at RT.
2. Add desired volume of DNA (depending on ratio used). Flick tube to mix and sit 15 to 20 minutes at RT.
3. Add transfection mixture to plate (make sure you gently swirl the plate to get even coverage).
4. Incubate at 37C O/N (or until cells are at desired confluency).
Our lab generally does not change the media the following day, since we use the cells in 24 hours or less...hope this can help!
I use the CHO-K1 line and did much optimization for transient transfection. I've never gotten Fugene6 to work well in CHO cells. I find that lipofectamine2000 is much better for this cell line.
I agree with drbrgandmusic. I also do transient transfection with FuGene6 that way (but I change the media next day).
Are your cells at proper confluency when you transfect them?
rkay447 might be right, too. Try another method/reagent to transfect your cells to see if that helps.
Good luck
Thanks guys! I really appreciate the help!
The cells are definitely confluent when I transfect them. I was trying not to use Lipofectamine 2000 as it requires the wash steps prior to adding the transfection mixture. And doing that on MANY 6 -well plates or even 96 well plates would not be all that fun.
But, it is possible that using Lipofectamine 2000 would be the better way.
I'm going to try again tomorrow-- I'll let you know how it all comes out.
Thanks again!
Are your cells at proper confluency when you transfect them?
rkay447 might be right, too. Try another method/reagent to transfect your cells to see if that helps.
Good luck
Hey Zek,
When I transfect, I usually have 3.75 x 10^4 cells per mL.... this is being done in a 6-well plate. When you transfect, what's your confluency?
Are your cells at proper confluency when you transfect them?
rkay447 might be right, too. Try another method/reagent to transfect your cells to see if that helps.
Good luck
Hey Zek,
When I transfect, I usually have 3.75 x 10^4 cells per mL.... this is being done in a 6-well plate. When you transfect, what's your confluency?
As far as I know, the cell number depends on cell type. For transfection with FuGene6, it must be 50-70% on the day of transfection. Cells should not be clumped in the centre of the well etc. And for transfection with Lipofectamine, cells can be a more confluent (although I am not sure whether I am right).
The only requirements for lipofectamine2000 is media without antibiotics. I go ahead and split my cells the day before and seed in media without pen/strep. I also make sure they are seeded into the proper amount of media so the next day all I have to do is make the complexes and add it to the wells--no washes needed. Granted, working without pen/strep requires extreme care to avoid contamination but it's doable. Also, the only other major drawback to lipo2000 is the amount of DNA required. It's quite a bit more than what you use for Fugene.
p.s. you said that your cells were definately confluent when you transfect. Over-seeding will prevent a good transfection, especially with Fugene. Lipo2000 isn't quite as finicky about cell density and the instructions actually suggest 90% confluency compared to Fugene's 70%.