Using Lipofectamine - What kind of efficiency do you get? (Jan/25/2006 )

Hi All-

I've heard people claim some very high transfection efficiencies using lipofectamine 2000 (or brand X lipid reagent). I'm trying to express my proteins in NIH-3T3's, which I've heard are supposedly easy to transfect.

What is a typical transfection efficiency (in %) for any of you using lipid based reagents and what cell lines did you use?

What else might I optimize? I have tried changing DNA:Lipofectamine ratios, cell densities, transfections with an without serum. Any general tips on transfection in general??
Does anyone have good luck with another cell type? maybe I should try 293T's or something.


Thanks for any suggestions,

Mountainman

I get say 80-90% efficiency with lipofectamine. The trick is to make sure tha t hte cells are actively growing. One thing you could do is seed at a hiigh density (85-90%) in the evening and transfect it next morning. Dont wait for 24 h. Transfect it using the OptimMEM media and pull off the ttransfecting media after 5 h. Wash the cells and add fresh media and let it equilibrate for 18-24 h before dosing.

I use it for MCF-7 cells and transfect at around 200,000 cells/well. My eperiment typically lasts 36-48h.

if u need a detailed protocol mail me back and I can send u one.
p..

Viability is sometimes the big issue with lipofectamine. It maybe an indication that you are transfecting too long or you have too high a concentration of DNA or reagent. What do your cells look like 24 hours post-transfection?

HI Again-


Pria, do you just do the DNA and LF2000 dilutions in OptiMEM, or do you culture the cells in it as well for the 5h transfection duration. I recently tried just diluting in OptiMEM (rather than DMEM) and adding the complexes to my cells in normal DMEM based media and may have seen a marginal increase in efficiency (my method is subjective as it's Fluorescence microscopy). Do most cell lines you work with seem to respond ok to OptiMEM for the transfection? When I tried to remove serum from my 3T3's in normal DMEM they came off the plate! (they seem to need serum for adherence)

Tap14, yeah there is a problem with viability which is a problem for making stables. It seems that the cells that are transfected also die (a lot of them). I think my DNA:LF2000 ratios are OK. I usually just leave the transfection complex containing medium on the cells for 24-48 hours though before I passage them. Should I try Pria's 5h reccomendation? Removing the complexes sooner would probably ameliorate my viability problem I suppose.

Thanks for the help, any other tips and questions are always appreciated too

Mountainman

Yeah...I just dilute my DNA: lipo mixture in OptiMeM and add it to my DMEM ....for eg. I seed cells in 500 ul DMEM. Next day i just make up my mixture such that each well gets 100 ul of the transfection liquid (DNA: lipo in Optimem).. Keep this for 5h and then pull off all the media at the end of 5h.
4-6h is more than adequate for lipo to transfect. If you keep it for longer the cells will definitely die. Been there....done that

Also serum is extremely important. The poor babies are already shocked with the us forcing the new DNA into them...u dont want to starve the babies. You need the cells to be actively growing so that they can take up the DNA. Serum starvation just puts them into the Go/g1 phase and u will got get much transfection.

As for ratios I use 0.5-0.8 ug DNA in each well (24-well plate) and use 1.5-2.0 ul lipo/ well. Another thing that may make a differnce is the way u mix the tubes. Add the DNA mixture to the lipo mixture. Not the other way around. I know for some transfection agents (eg JetPEI) the way the mixture is made makes a difference.

Secondly, add the lipofectamine to a tube which already contains the optimem. i.e do not add lipo to an empty tube. I just do it this way since another reagent called fugene loses its transfection efficiency when it comes in contact with plastic. They ask yo to add it to a tube already containing some liquid. Just being cautious thatsalll....but (knock on wood) it works for me. good luck....

lemme know if u need anything else....
p..

Ok thanks Pria!!!

I'll try using OptiMEM and adding the DNA mixture to the LF2000 mixture. And of course I'll try removing the transfection complexes after 5h. Will simply adding the DNA mixture to the LF mixture (rather than my usual vice versa) and using optiMEM increase my efficiency significantly? Right now I must be operating around 5-10%, which isn't very good.

I figure there must be something else subtle I'm missing because 5-10% isn't very good. I usually pipette the mixtures carefully, as I've hear they're delicate. Are there any other subtle technical elements I might be forgetting?

Anyways, I'll post again around next Tues/Weds when I try another transfection and let you know if my efficiency has improved.

Thanks for all the help,

Mountainman

Adding the DNA to lipo might affect the transfection. It does so for another transfection agent called jetPEI.... I just am a little cautious.

Another thing that also helps is to make sure everything is at room temp. ie the lipo and the optimem. incubate the mixture for 20 mins beofre adding it to cells. Also adding the transfection solution drop by drop in the centre of the well helps too...

I am assuming that ur plasmid is good and has the 260:280 ratio between 1.8-2.0. If so ...i am positive that the above tips will help u in getting ur transfection efficiency.

good luck
p...