Invitrogen’s Lipofectamine 2000 is one of many cationic lipid-based transfection reagents presently on the market. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Invitrogen claims that this reagent gives the highest protein expression levels in the widest variety of adherent and suspension mammalian cell lines compared to similar products. It has also been shown to work well with some primary cell lines and is one of the most widely used transfection reagents for use with siRNA. Transfection conditions can be easily established using Invitrogen’s application protocols. In addition, Invitrogen maintains a database of cell lines at www.invitrogen.com/celllines with valuable information about specific cell lines.
Lipofectamine 2000 is an easy to use transfection reagent. It is recommended that cells are growing at 90-95% confluence prior to transfection (for adherent cells). The media needs to be changed at least 1hr prior to transfection. A nice feature is that transfection can be performed in the presence of serum so no need to have many different types of media on hand and very useful for primary cell lines. Both Lipofectamine 2000 and DNA are mixed with Opti-MEM media and the quantity of each is dependant on the type of culture vessel (i.e. flask, Petri, 12 or 96 well plate). After a 5 minute incubation in OPTI-MEM, the Lipofectamine 2000 and DNA are combined and incubated for 20 minutes with gentle shaking at room temperature. The DNA:Lipofectamine 2000 complexes are then added to the cells, the media is swirled to ensure even coverage and the cells are incubated overnight. Due to the low toxicity of Lipofectamine 2000 there is no need to remove the complexes after incubation, a drawback of many other transfection reagents, and cells can be assayed for protein expression after 24hrs. In the case of transfection of stable transformants, cells are passaged at a 1:10 dilution after 24hrs and the media is changed to selective medium after 48hrs. Invitrogen recommends that antibiotics should not be present in the media during the transfection, as this will cause cell toxicity.
We have been using Lipofectamine for more than 2 years and it is probably the most simple and efficient transfection reagent available today and certainly our transfection reagent of choice. We use it regularly for a range of cell lines such as HEK-293, CHO and COS-7 and obtain consistently greater than 90% efficiency. More impressive is the performance of Lipofectamine with primary cell lines, which are notoriously difficult to transfect. Transfection of primary synoviocytes resulted in greater than 50% transfection efficiency. We have also used Lipofectamine 2000 to introduce siRNA into primary synoviocytes and obtained a comparable transfection efficiency when transfecting larger DNA species (Scott et. al. BBRC 328 2005 pp409-414). Invitrogen’s recommendations are best followed closely for transient transfections but we have noticed that in HEK-293 cells transfected for stable transformants there is a degree of flexibility in these recommendations. Cell densities of less than 90% and up to 100% confluence have worked well and the presence of antibiotics in the transfection media did not affect the end result. In the two years we have used Lipofectamine 2000 for transfection reactions, when performing transfections for stable transformants, we have yet to have an unsuccessful transfection.
Boyd Scott
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals