transfection of NRK cells-lipofectamine toxicity - (Sep/06/2006 )
I am desperately trying to transfect NRK cells. I need high efficiency of transfection in order to do western blot on transfected cell lysates. Unfortunately, reagenst like fugene6 and TransLT-1 give only 5-10% efficiency. Lipofectamine gives 70% positive cells, but seems to be toxic. The cells look changed in morphology and sick. Since I need to assess changes in membrane organisation for my expt, this doesn't work ofr me.
Does anyone know how to reduce toxicity effect with lipofectamine and/or know of any other transfection raegent that will give more than 50% efficiency in NRK cells?
How do you transfect your cells with Lipofectamine? We add the DNA-lipid complexes to the cells in complete medium (serum, antibiotics) and leave them for 24 hours. After 24 hours we lyse the cells. An alternative protocol is to remove the full medium from the cells, add serum free medium, DNA-lipid complexes for 4 hours, replace the medium with full medium. One of the two should work. I find the first has lower toxicity in my case. The alternative is to try Lipofectamine LTX, it is new and you can ask Invitrogen for a free sample (we got it but we haven't tried it yet). It is supposed to have low toxicity.
u should try dnafactory's suggestions.
I have also found that increasing cell numbers can reduce toxicity and also low passaged cells r more tolerant.
I have also found that increasing cell numbers can reduce toxicity and also low passaged cells r more tolerant.
Thank you scolix!
Does anyone know how to reduce toxicity effect with lipofectamine and/or know of any other transfection raegent that will give more than 50% efficiency in NRK cells?
You could also try Roche's new Fugene HD. It has higher efficiency than Fugene 6 without being toxic. Good Luck
How about the quality of your DNA prep? Certain cells are more sensitive to endotoxins (U87 for instance, an astrocyte I'm working with) than others (293 for instance).
Typical for endotoxins was that my cells transfected nicely (checked by EGFP expression in my case) but instead of remaining adherent and growing nicely afterwards they floated around and morphology changed too.
Typical for endotoxins was that my cells transfected nicely (checked by EGFP expression in my case) but instead of remaining adherent and growing nicely afterwards they floated around and morphology changed too.
That's really I good point!
Lotus, maybe you should try another kit for DNA prep, that can give you endotoxin free DNA
lotusgirl: Are you trying to tell us different stories in different posts? First, you told us you are not getting good result with IT (10% transfection rate) in this post, however, minutes ago you said IT did great job for you. See below for details in your previous post. In fact you did not even tell the same story at the begining (much lower price) and at the end (cost mounts up to the same level) in this one post. Whats going on?
I have beed regularly using Transit LT-1 from Mirus. It is GREAT! Works much better than Fugene6, at much lower price and less toxixcity to cells. I use NRK cells which are difficult to transfect . Instead of fugene6, I tried Mirus lt-1 I was amazed it worked much better than Fugene6 when I followed their recommended protocol. Mirus usually gives free sample so you cana sk for it and try on your cell line first.
Only drawback. They recommend 7.5 microlitres of the reagent per 35 mm dish per transfection. that means we use more of it per transfection so in the ned the cost mounts up to the same level as of fugene and lipofectamine.
I have beed regularly using Transit LT-1 from Mirus. It is GREAT! Works much better than Fugene6, at much lower price and less toxixcity to cells. I use NRK cells which are difficult to transfect . Instead of fugene6, I tried Mirus lt-1 I was amazed it worked much better than Fugene6 when I followed their recommended protocol. Mirus usually gives free sample so you cana sk for it and try on your cell line first.
Only drawback. They recommend 7.5 microlitres of the reagent per 35 mm dish per transfection. that means we use more of it per transfection so in the ned the cost mounts up to the same level as of fugene and lipofectamine.
u r a good detective, genehunter
I have beed regularly using Transit LT-1 from Mirus. It is GREAT! Works much better than Fugene6, at much lower price and less toxixcity to cells. I use NRK cells which are difficult to transfect . Instead of fugene6, I tried Mirus lt-1 I was amazed it worked much better than Fugene6 when I followed their recommended protocol. Mirus usually gives free sample so you cana sk for it and try on your cell line first.
Only drawback. They recommend 7.5 microlitres of the reagent per 35 mm dish per transfection. that means we use more of it per transfection so in the ned the cost mounts up to the same level as of fugene and lipofectamine.
That's not good... Thank you genehunter-1 for finding out...