Hey, Easy On Those Cells!

Cells transfected using FuGENE 6 reagent and protocol supplied by Roche Molecular Biochemicals. MCF-10A cells transfected with a GFP construct (pGreenLanternn-1; Gibco/BRL) and fixed with formaldehyde and counter-stained with DAPI to reveal nuclei. GFP expression (green) was detected by fluorescence microscopy using a standard FITC filter set. DAPI staining (various shades of blue) was detected by fluorescence microscopy using a DAPI filter set. Transfected cells have a light blue (almost white) nucleus surrounded by green; the lighter blue (white) is due to an overlap in the two colors. Image submitted by Randall Marsh, University of Cincinnati College of Medicine. Your transfections aren't working. Maybe your current method kills most of your cells. Or maybe you're working with a reluctant cell line and want to boost your transfection efficiencies. Not only have you attempted the various methods available, but you've tried every trick in the book. Maybe it's time to give FuGENE 6 Transfection Reagent a try. After speaking with some researchers and viewing testimonials on the Roche web site, it is clear that this reagent has proved to be the answer to many a transfection problem. FuGENE 6 reagent is an optimized blend of lipids and other proprietary components that permits high transfection efficiencies without the toxicity observed with some cationic liposome-based reagents. Enabling both transient and stable transfections, FuGENE 6 reagent is simple to use; users mix the plasmid of interest with FuGENE 6 reagent and add the mixture to the cells in culture. No additional manipulation is necessary since the reagent functions equally well in the presence or absence of serum. And in almost all applications, as a result of its low cytotoxicity, FuGENE 6 does not need to be removed prior to performing a reporter gene assay. The product is supplied, ready for use, as an 80% ethanol solution. Aliquots can be repeatedly removed from the sample vial with no loss of efficiency over several months. For users' convenience, Roche Molecular Biochemicals (formerly Boehringer Mannheim Biochemicals) maintains a regularly updated list of cell types on its web site biochem.boehringer-mannheim.com/techserv/fugcell.htm) that independent researchers have successfully transfected with FuGENE 6 reagent. At the time of this writing, FuGENE 6 had successfully transfected over 120 different cell types. These range from the standard COS-1 and HEK-293 cells to primary human skin fibroblasts and rat tracheal epithelial cells. Also listed are cell lines that some labs (but not all labs) have successfully transfected (5 cell lines), and to avoid wasting precious lab time, Roche lists cell types that researchers have failed to transfect with the reagent (13 cell lines). FuGENE 6 reagent proved its usefulness to Sumiko Kurachi, a research scientist at the University of Michigan. Kurachi had been working with the Hep-G2 (human liver hepatoblastoma) cell line with some significant difficulties in achieving a high transfection efficiency. Every transfection reagent Kurachi had tried yielded only poor results, while calcium phosphate-mediated transfection had allowed only five to seven percent efficiency. With FuGENE 6 reagent, Kurachi was able to achieve much higher transfection efficiencies with Hep-G2 more easily and with greater reproducibility. Recently, Kurachi was able to obtain stable transfectants with Hep-G2 using FuGENE 6 transfection reagent. Similarly, FuGENE 6 was the answer to Ben Williger's transfection problems. Williger, a research associate at Vanderbilt University (Nashville, Tenn.) had previously transfected HT-1080 human fibrosarcoma cells with a lipid-based transfection reagent with limited success. He then switched to FuGENE 6 and achieved successful transfections with expression levels several fold higher than observed with his previous method. FuGENE is a trademark of Fugent, L.L.C. For more information contact Roche Molecular Biochemicals at (800) 428-5433 or visit the FuGENE 6 Transfection Reagent web page at http://biochem.boehringer-mannheim.com/techserv/fugene.htm The author can be reached online at sbeck@the-scientist.com.

Hey, Easy On Those Cells!

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