Lipofectamine™ 2000 is a lipid based transfection reagent for eukaryotic cells. It can be used for transfection of adherent or suspension cells with DNA or RNA (including Stealth RNAi and siRNA) and can be used for either transient transfections or to create stable cell lines. The reagent can be used in the presence of serum, and the reagent does not need to be removed from cells. It is supplied as a sterile liquid in volumes of 0.75 ml, 1 ml and 1.5 ml. It is stored at 4C and is guaranteed stable for 6 months if stored correctly.
Using the transfection reagent is simple, however the success of its use is dependant on maintaining a healthy, low passage cell line and good tissue culture technique. To begin, the cells are seeded in tissue culture plates so that they are 90-75% confluent at the time of transfection. This is usually done either the day before, or for suspension cells directly before the transfection. The Lipofectamine™ 2000 and the DNA are diluted in separate polypropylene tubes with a small volume of serum free media. After a 5 minute incubation, the two volumes are mixed, and the tubes incubated at room temperature for 20 minutes to allow the reagent-DNA complexes to form. The complexes are added directly to the media already on the cells, and the plates are incubated for the desired amount of time. The media may be changed after 5-6 hours to prevent toxic effects. The product manual includes advice on how to optimize transfection efficiency, as a number of conditions can be varied including DNA concentration, amount of transfection reagent and cell number at the time of transfection. The manual also includes guidelines for scaling transfections up or down, e.g. from 6-well to 96-well plates.
A number of factors affect this reagent, which must be considered before purchasing. Lipofectamine™ 2000 is designed to be used with Invitrogen media with the manufacturers recommending OPTI-MEM for best DNA-reagent complex formation. Other media which may be used (but apparently less efficiently) include DMEM, RPMI 1940 and PBS. In terms of the cell line, the manufacturers have tested the reagent in a number of lines including several standard mammalian lines (VERO, 293H, COS-1, CHO) and found it to transfect at 80-99% efficiency. It is worth checking their website before starting, as they have a number of protocols provided for standard cell lines.
We have used Lipofectamine™ 2000 for several years in our laboratory on a variety of mammalian cell lines (VERO, COS, PS/EK, BHK), using Invitrogen media and DNA vectors. Transfection efficiency varies depending on the cell line, but we generally get around 50-80% transfection efficiency. We have found that the success of the transfection is greatly affected by the seeding density of cells. We tend to follow the protocol provided, which is simple and easy to follow. We have found that it is necessary to change the media after 5-6 hours to prevent toxic effect on the cells, despite this being an optional step.