Lipofectamine 2000 (Invitrogen) - How much can transient transfection expected ? (Oct/27/2005 )
I just got my HepG2 cells fro ATCC and it is in Passage-4 which i have want to freeze.
Kindly let me know the freezing medium ?
Latter i have planned to do transfection experimetns using Lipofectamine in 24 well plate.
So any one who has done can tell me the efficiency of transfection using Lipofectamine.
If any suggestions and precaution to be taken. please make be aware.
I don't know these cells (so I can't help you out on the freezing medium), but for the transfrection efficiency: that depends on your cells themselves, on the quantitiy of DNA and on the quality. I performed lipofectamine2000 transfection on HELA cells and got really good results (Checked transient transfection under fluorescence microscope and had very high efficiency). Used the same reagent and methods (including wash steps) and plasmid on U87 cells and had very bad results (high toxicity with normally adherent cells in suspension).
About precautions: make sure to get endotoxin free plasmid DNA, and if you're not too sure about the sensitivity of your cells: remove the lipofectamine2000 + DNA after 3-4 hours and wash your cells before adding fresh medium.
I have some experience with HepG2 cell line. The freezing medium is the medium what you normally use to culture the cells, plus 5% DMSO. Anyway you can check the information in ATCC pages about this line's requirements, to make sure.
I do not know exactly the eficiency of transfection with Lipofectamin 2000, I haven't tried transient transfection, but I tried stable and I can say it was "very low", I got clones but quite few compared with the total amount of cells.
Now i am working on HepG2 cells. About cell stock media, you can use formula as same as normal cells. In my case, i followed this formula:
SFM 60% + FBS 30% + DMSO 10%
About transient transfection in HepG2 cells, I strongly recommend you using Metafectene reagent. Its transfection efficiency is around 70-90% in HepG2 cells as well as Vero, Hela...
Goodluck to your work!!!!
Thank u very much