If working with nucleic acid transfections in cell culture, consider Cellfectin® Reagent from Invitrogen. This reagent utilizes cationic lipid transfection technology for the transfection of DNA into insect or mammalian cells (adherent and suspension). The reagent has been specifically designed for transfecting insect cells, in particular DNA from the BaculoDirect™ or InsectSelect™ expression systems into Sf9, S2, Sf21 and High Five™ cells. The reagent is supplied as a single 1 ml aliquot, which can be stored at 4ºC for up to 6 months.
The protocol for using the reagent is similar to other transfection products – the following is an outline of the procedure for transfecting baculovirus DNA into Sf9 cells in a 6 well plate. To begin, plate out 9 x105 cells in 2 ml media and allow them to attach for an hour. Meanwhile, add 1-2 µg DNA to serum-free media for a final volume of 100 µl. In a separate tube, mix 1.5 – 9 µl Cellfectin® Reagent in another 100 µl media. The two solutions can then be mixed, and complexes allowed to form for 15-45 minutes; 800 µl serum-free media is then added to the tube. The cells are washed once with serum-free media, then this is replaced with the transfection mix. To allow transfection, cells are usually incubated for 5 hours, after which the media is replaced with normal growth media and incubated for the desired amount of time. The manual supplied with the reagent also includes protocols for both suspension and adherent mammalian cells, which are similar to the protocol outlined above.
There are some general considerations for transfections using the Cellfectin® Reagent. As with all cell culture experiments, transfections should only be performed on healthy, low passage cells for best results. No antibiotics can be used during transfection as this leads to cell death. The manufacturers state that serum-free media should be used to form the DNA/reagent complexes, and recommend that serum-free media is used during transfection, although this is not essential. The reagent is designed to work best with Invitrogen media such as Sf-900 SFM and OPTI-MEM®. Other media should be tested in case they inhibit transfection. As with any new transfection reagent, the user must optimize cell density, DNA and Cellfectin® concentrations and time of expression for highest efficiency transfections. Unlike other lipid based reagents, no mention is made in the manual of needing polypropylene tubes for forming the complexes.
We have been using the Cellfectin® Reagent in our lab for quite some time for transfecting Sf9 and High Five™ cells, mainly baculovirus DNA and occasionally the pMIB/V5-His vectors from Invitrogen’s InsectSelect™ system. For these constructs, transfection resulted in high levels of both virus and protein being produced. Minimal optimization was required, a bonus of using a transfection reagent designed for a specific cell line and system. We used Sf-900 SFM (Invitrogen) for all transfections. We have also used the reagent on another insect cell line (C6/36); however, this cell line required more optimization and adaptation to new media before decent levels of transfection were seen.
Research Scholar
School of Molecular and Microbial Sciences
The University of Queensland