I have been transfecting HEK293 cells successfully with a GFP fusion protein using Lipofectamine 2000. I optimised the transfection as suggested by the manufacturer and found them to be successful at all ranges tested (Lipofectamine:DNA ratios of between 1:0.5 and 1:5). The optimum happened to be around the manufacturer's suggestion of 4 ug DNA and 10 ul lipofectamine in a well of a 6-well plates but in all cases there was acceptable transfection efficiency and no noticeable cell death 48 hours post-transfection.
I've then performed further transfections with which to select stable cell lines with no appreciable cell death until i started performing selection.
A few (maybe 5 or 6) passages later I've been trying to repeat my transfection under the optimised conditions in order to perform some experiments but when i come to look at the transfections the next day they're dying. By 48 hours almost all cells are floating. This is true when performing the transfection in flasks or on poly-d-lysine coated coverslips in 6-well plates.
My colleague has found the same thing - she has been passaging the cells independently, transfecting with a plasmid encoding a different protein and using a different tube of lipofectamine. While her transfections worked initially she now also observes cell death 24 hrs post transfection.
In both cases there is no sign of contamination even after leaving the flasks/plates in the incubator for a further week or so.
So, we don't believe the DNA and the lipofectamine are the problem as both have performed fine previously and we''re sure we're not getting infections. We've even tested the FCS we're using because we have recently changed supplier. We are at a loss and can only think of thawing another vial and seeing if those cells will transfect ok.
Can anyone think of any possible reasons for this? My colleague and I are getting very stressed out over this holding up our work...
Thanks for reading,
Edited by gantzgraf, 29 July 2009 - 02:36 AM.