Problem with expression of marker gene - (Sep/02/2005 )

Hi all...
I'm new in this field and I have some problem...

I infect 3t3 cells with lentivirus sups that has GFP marker gene to find out if lentivirus work or not.

I use 6 well plate that already had 3t3 cells ready for infection and I use 3 different volumes of the lentivirus sups 500,250 and 100 microlitter and I bring up to 500 microlitter with DMEM..
For the positive control I use leftover lentivirus sups(already in -70 for 3 weeks) that already tested for expressing GFP in the infected cells before.
For negative control I only put DMEM at 3t3 cells.

after that I put 0.8microlitter polybrene and incubate at 37 degree for 5 hour
and put DMEM again and incubate again for 2 days.

After 2 days I aspirate and wash with pbs and trypsin and put hbss, spin them and resuspend pellet with hbss again..
After that I test them with FACS and the results are all the samples did not have GFP expressed. All of them looked the same as negative control in FACS..

I guessing maybe the infection step that went wrong because my positive control not worked too but I'm not sure about this...
I 'm so confused what went wrong ...is anybody know why they not expressing GFP?

Thanks in advanced...

I think 2 days probably isn't enough, let them go longer .... think about what has to be done: the cells need to be transduced, the RNA must be made into DNA and then incorporate into the genome and then be expressed. It's a lot to do in just 48 hours!

I usually check my lenti infections at 4 days atfer transduction.

Other thoughts:
- What concentration polybrene are you using in ug/ml??
- Are your cells healthy, fresh and mycoplasma free?
- What promoter are you using?
- Maybe you should leave the lenti on for longer - say 6-16 hours before diluting? And then remove the polybrene and replace with fresh media ... cells don't "like" polybrene.
- Try concentrating the virus with high speed centrifugation.
- Also and importantly ... perhaps you should PERMEABILIZE your cells in case your flow cytometer isn't detecting the GFP inside the cell?
- Have you looked at your cells with a fluorescence microscope to see if you see the GFP? If not, let them go longer!!