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December 01, 2010 (Vol. 30, No. 21)

Utilizing FuGENE to Optimize Transfection

A Few Key Considerations Are Essential to Obtain the Best Return from Cell-Based Experiments


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    Figure 2. FuGENE HD transfection optimization experiment: (A) Layout for a 96-well plate to determine optimal transfection conditions for FuGENE HD Transfection Reagent. Transfection mixes (100 µL) as indicated were prepared in a separate 96-well plate. Transfection mixes were transferred to the transfection plate after a 15-minute incubation (RT). All test wells contained cells in growth medium. Column 2: Control consisted of medium (10 µL), DNA (10 µL, 20 ng/µL), or FuGENE HD Reagent (10 µL). Columns 3–11: Transfection mix with FuGENE HD Reagent and DNA as indicated. (B) Timeline of the optimization experiment. Day 1, plate is seeded with medium or healthy cells (100 µL). Day 2, controls and DNA:FuGENE HD Reagent transfection mixes are prepared and added to cells. Day 3 or 4, cell viability and reporter activity are assayed.

    Optimal transfection conditions for any cell line should be determined empirically. It is worthwhile to spend the time up front to ensure maximal response from the cells in all subsequent experiments. The FuGENE HD Transfection Reagent is lipid-based, simple-to-use, and can result in high transfection efficiencies with minimal cyototoxicity. Figure 2 presents an example of optimized transfection conditions for the FuGENE HD Reagent. Test variables include the ratio of reagent:DNA and volume of transfection mix added.

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    Figure 3. HEK293 transfection optimization experiment: Optimal transfection conditions for HEK293 cells with FuGENE HD Reagent were determined using the protocol outlined in Figure 2. Cells were grown to 85% confluency, harvested, and added to a 96-well plate at 2 x 104 cells/100 µL/well. The next day, the pGL4.13 Vector expressing firefly luciferase was diluted in serum-free medium (DMEM, 100 µL) to 20 ng/µL and mixed with FuGENE HD Reagent to achieve the indicated reagent:DNA ratio. After a 15-minute incubation, indicated volumes were added per well. The cells were mixed gently and incubated for 24 hours at 37ºC/5% CO2. Cell viability (CellTiter-Fluor™ Viability Assay) and reporter activity (ONE-Glo Luciferase Assay System) were assessed as in Figure 1. Data are the average of replicate samples ± SEM.

    The FuGENE HD volume-to-DNA mass ratio (µL/µg) determines the charge of the mix added to the cells (the negatively charged DNA must be balanced by the positively charged, cationic lipid of the reagent), and the volume of this mixture determines how much DNA is administered. More is not necessarily better and may lead to reduced protein expression and reduced cell health (Figure 3). Typical reagent:DNA ratios are between 1.5:1 and 4:1 with addition of 2–10 µL per well. In this experiment, optimal conditions for HEK293 transfection were 5 µL of a 2.5:1 mix.

    Controls should be included in optimization experiments. Untransfected cells are used as an indication of maximum viability and no reporter expression; DNA- and reagent-only controls are included to monitor any unexpected effects of the transfection mix components on the cells.

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