When are the cells virus free? - (Jan/28/2007 )
After doing lentiviral infection of the cells if I need the cells for other experiments(say FACS) when can we consider the cells to be virusfree? After two trypsinizations or anything like that? Thanks
The lentiviral particles are inactivated within a few hours (8-12 hrs) in media. And I would normally change media 12 hrs after infection so the cell would be virtually free of virus after 12 hrs. I know people use infected cells after 2 days for FACS to determine titres
centrifugation does not pellet viruses.
i know that half life of my retrovirus is 6h in medium.
So 12h after infection plus pelleting should ensure to get rid of viruses.
Sorry, I don't understand how this can be...when we produce lentivirus in 293FT cells they are sitting in medium for at least 48 (and up to 72) hours until we harvest the medium and freeze down. These viruses are able to transduce cells, so this means they weren't inactivated in the medium (when being produced? Am I forgetting something very simple here? I just can't see any difference between 293 medium and medium used when transducing cells (sure, composition is different, but both are essentially growth medium containing serum).
If I understand you're remark correct the clue is that you're 293FT cells are continuously producing virus particles, so the first ones (produced during maybe the first 24 hours) will indeed be inactive (non-infectious) but the ones produces after for instance 40 hours will be active at 48 hours (harvesting time).
I was describing infection of lentivirus and not production.
During production, they are continually being produced. And the best titres are if collected at 24-36 hours after infection. The virus particles have a half life of 8-12 hrs so Ideally one would collect virus every 12 hrs, I collect every 24 hrs and my first collection is 3x better than second collection.
Ah, that makes sense and is great information for the future, I'll definitely harvest the viruses at 48 hours and not wait until 72 now!