5 replies to this topic

[hhhhyyyy]

I have adenoviral vectors with my gene of interest in them.  I have a problem about Viral production in 293 cells.
After I transfer 293 cells with lipofectamine 2000 and Pac I digested  Vector, almost all of 293 cells float.
The follow is my protocol:

1.  plate cell in 6 well plate with  DMEM without antibiotics. After 24h,cells reached 90% confluent.

2. prepare DNA-LF2000 mixture strictly according to manufacture`s manual. 4uL DNA and 10uL pofectamine2000 was added per well.

3.  remove  cells medium with  2mL serum free DMEM  and add DNA-LF 2000 complexes gently.
4.  After incubate for 6h, chang medium with fresh complete DMEM.But after 12h all of cells had float.

how can I decrease  the LF2000 toxic effect and modify the protocol?

thanks a lot:)

Edited by hhhhyyyy, 03 August 2004 - 06:46 AM.

[kant0008]

Hi,
I probably misunderstood you, but are your cells in serum-free media for 6 hours? I actually keep my cells with serum in media at all times, serum free media is only required for incubation of Lipofectamine and plasmids. So I don't change media before I transfect, just add Lipo with plasmid in serum-free media to ready wells.
Also you could decrease the concentration of Lipo, I find 5ul per well works fine.

[hhhhyyyy]

Thanks for your reply.
I will follow your opinion. The manufacture`s manual also indicates it is not necessary to use serum free DMEM during 6h transfection. But I have some other questions to ask you.

1.  One day before transfection, how many cells do you plate in per well plate.Do you count it?

2. Your lipo is 5uL, but how many viral plasmid do you use? Is it  2ug?

3. Is it necessary to remove the DAN-LF2000 complexes and chang medium after 6h ? which time will better to chang medium? 4h or 6h or 24h?

[kant0008]

Hi again,
about your questions:
yes, 2ug vector works well, with 5ul Lipo.
I normally plate about 1X10.5- 5X10.5 cells per well of a 6well plate, but it's different for different cells, just go with the number that will give you 90% confluence after 24hrs. And yes, I do count.
Again I personally don't bother changing the medium after transfection, but my cells are quite robust and if you are having problems with toxicity I'd change the medium, 6hours is probably best.
Hope this helps

[hhhhyyyy]

thanks  for your suggestion

[CaP]

Hi,

Hope you still watch this.
I've been doing a lot of those.
What I felt was, maybe you should obtain a "new" 293 cells rather than changing your protocol.
Sometimes transfection or plaque assay are critically dependent of cells.  If they look pretty tiny and easier to come off, you won't get good result.  So called "low passage" of 293 is preferable if available.  Ask around in your institute who does a lot of adeno.

One more thing, I tried Lipofectamine 2000 and PolyFect (Qiagen) at the same time, with the same DNA.
Believe or not, I got a lot of CPE much earlier with PolyFect than Lipo2000.  I got CPE from Lipo2000 eventually, but it was like a week after (I got CPE ~10days with PolyFect).

Good luck,