Lipofectamine 2000 Reagent is a cationic lipid-based transfection reagent designed to give extremely high transfection efficiencies and result in equally high protein expression levels. It has also been optimized to work for many mammalian cell lines. Invitrogen already markets several transfection reagents and so to produce another you have to expect improvements over their existing products, as well as over competing company’s reagents.
So far, I have been very impressed by the performance of Lipofectamine 2000. It is very easy to use, somewhere between Lipofectamine and FuGENE 6! An appropriate amount of the cationic lipid is mixed with Opti-MEM, ~4 ul of reagent with 50 ul of medium per well of a 12-well plate, and incubated for 5 minutes at room temperature. The DNA for transfection, 1-2 ug per well of a 12-well plate, is added to another 50 ul of Opti-MEM. After the first 5 min incubation, the diluted DNA is added to the diluted reagent and a further 20 min incubation at room temperature is performed to allow DNA-lipid complexation. DNA-Lipofectamine 2000 complexes are then added to cells, in 100 ul aliquots for 12-well plates, and cells are returned to their incubator after a gentle mix. It is not necessary to remove the complexes and as such transfections can be completed in around 30 minutes. The reagent can be used on adherent or suspension cells and scaling up or down is easy by changing volumes of DNA, reagent and Opti-MEM. Finally, because of its low cytotoxicity, Lipofectamine 2000 can be used to transfect cells during initial plating which may be applicable to high through put formats.
More important than ease of use is success of transfections. With Lipofectamine 2000 I have been able to transfect an epithelial cell line with good efficiency that I previously struggled with. Also, transfection efficiency in some of the fibroblasts I work with is higher with Lipofectamine 2000 than the previous reagent I used ? this is, however, somewhat redundant as for my purposes the older reagents gave sufficient expression and efficiency. Another plus about Lipofectamine 2000 is that it is comparably priced to other reagents so that there is no extra cost for the increased performance. The only minor issue I have with Lipofectamine 2000 is that transfection needs to be performed in the absence of antibiotics. This should not be an issue with antibiotics that are used as a precaution, such as penicillin and streptomycin, however, it may add an extra step (of changing medium) if a transfection is being performed in a clonal cell line that has previously been selected by growth in an antibiotic.
Peter Haggie, Ph.D.
Post-Doctoral Fellow
University of California, San Francisco