RAW 264.7 cell transfection - Probs transfecting RAW cells with lipofectamine LTX (Mar/08/2008 )
Hello.
I'm having problems transfecting my RAW cells using lipofectamine or lipofectamine LTX. I got it to work very well before Christmas but it hasn't worked since because the transfection efficiency has been very low.
Lipofectamine method: Plate 2x10^6 cells per well in a 6-well plate in DMEM+FCS+supps+antibiotics. One hour before transfection, wash cells with Optimem and then add 2mls Optimem media. Per well, I use 3ul lipofectamine, 1ug luciferase plasmid adn 1ug renilla. I also add 1ul 1mg/ml proteamine sulphate since (I came across a paper where this was used and it enhanced transfection). After 5hrs, I change the media to DMEM+FCS+supps+antibiotics and add cytokines o/n. I then measure luminescence the next day.
The protocol for LTX is similar: I use 6ul LTX.
Any help will be appreciated, thanks!
TKK
I'm having problems transfecting my RAW cells using lipofectamine or lipofectamine LTX. I got it to work very well before Christmas but it hasn't worked since because the transfection efficiency has been very low.
Lipofectamine method: Plate 2x10^6 cells per well in a 6-well plate in DMEM+FCS+supps+antibiotics. One hour before transfection, wash cells with Optimem and then add 2mls Optimem media. Per well, I use 3ul lipofectamine, 1ug luciferase plasmid adn 1ug renilla. I also add 1ul 1mg/ml proteamine sulphate since (I came across a paper where this was used and it enhanced transfection). After 5hrs, I change the media to DMEM+FCS+supps+antibiotics and add cytokines o/n. I then measure luminescence the next day.
The protocol for LTX is similar: I use 6ul LTX.
Any help will be appreciated, thanks!
TKK
It appears that your DNA to reagent ratio is little off. I would use 1: 4-6 ratio. eventhough the transfection rate is far less than other cell types.
Protamine enhances some but not all transfection agents like lipofactamine.
I'm having problems transfecting my RAW cells using lipofectamine or lipofectamine LTX. I got it to work very well before Christmas but it hasn't worked since because the transfection efficiency has been very low.
Lipofectamine method: Plate 2x10^6 cells per well in a 6-well plate in DMEM+FCS+supps+antibiotics. One hour before transfection, wash cells with Optimem and then add 2mls Optimem media. Per well, I use 3ul lipofectamine, 1ug luciferase plasmid adn 1ug renilla. I also add 1ul 1mg/ml proteamine sulphate since (I came across a paper where this was used and it enhanced transfection). After 5hrs, I change the media to DMEM+FCS+supps+antibiotics and add cytokines o/n. I then measure luminescence the next day.
The protocol for LTX is similar: I use 6ul LTX.
Any help will be appreciated, thanks!
TKK
It appears that your DNA to reagent ratio is little off. I would use 1: 4-6 ratio. eventhough the transfection rate is far less than other cell types.
Protamine enhances some but not all transfection agents like lipofactamine.
I'll repeat the transfections with a higher ratio, thanks.