was rigorously disinfected, a new source of HeLa cells was used for another series of optimization studies. These cells showed the expected, standard optimization profile and showed no contamination, as detected by the Mycoplasma PCR Primer Set (Figure 3, lane 3).

Comparing Lipid-Based Formulations

In most cases, researchers desire to define transfection conditions that produce the highest possible number of transfected cells for data analysis. For this reason, researchers want to use the reagent that reliably yields the highest transfection efficiencies for their cell lines. Several studies confirm that efficiencies vary for different lipid-mediated transfection reagents among cell types.⁵

One research group used an experimental system consisting of transient transfections of the human adrenalcarcinoma cell line (SW13), with a plasmid encoding the enhanced version of green fluorescent protein (EGFP). Calcium phosphate transfections yielded low transfection efficiencies and a corresponding subcellular localization of exogenous protein. To resolve these problems, they compared two liposome-based formulations, LipofectAMINE and LipoTAXI transfection reagents.⁴

The pEGFP-C3 plasmid, which encodes the EGFP with expression driven by the CMV promoter, and either LipofectAMINE or LipoTAXI reagent were used to transfect SW13 cells. The cells (8 x 10⁴) were plated in 24-well culture dishes and allowed to attach overnight to achieve 60% to 80% confluency. For each liposome-based product, the manufacturers recommendations were used to determine cell-culturing specifications and the appropriate range for reagent volume and concentration of the plasmid DNA. Following transfections, cells were fixed, coverslips were prepared, and fluorescent cells were counted.⁴

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