December 1, 2010 (Vol. 30, No. 21)

Trista Schagat
Kevin Kopish

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

Because optimal DNA delivery into mammalian cells depends on multiple factors, we recommend optimizing transfection conditions for all culture cell types. Optimization helps find the balance between maximal protein expression and minimal impact on cell viability. This article addresses the variables that should be tested when first optimizing transfection.


Successful Transfection

A number of factors will contribute to transfection success as well as the biological response of the transfected cells. We recommend careful consideration of each of the following parameters, and illustrate successful plasmid DNA transfections using FuGENE® HD Transfection Reagent from Promega.

Cell Health. Cells should be actively dividing, passaged regularly in fresh growth medium, and not allowed to become overconfluent at the time of transfection. Ideally, cells will be 75–90% confluent and greater than 95% viable just prior to transfection, and typically 80% confluent on the day of transfection using the FuGENE HD Transfection Reagent. Passage number should be monitored because the biological responsiveness of cells can vary at low or high passage numbers.

DNA Quality. Plasmid DNA used for transfections should be of high purity (A260/A280 of 1.7–1.9) with low endotoxin levels to avoid cytotoxicity or proinflammatory cytokine production. Use a method qualified to produce transfection-grade DNA.

Transfection Method. Lipid-based reagents tend to give the lowest toxicity and have been used to transfect a wide range of cell lines. Newer reagents such as FuGENE HD involve a single addition of DNA:lipid complexes to cells with no subsequent medium change. However, lipid reagents vary and the maximum protein expression and cell viability achieved can vary greatly (Figure 1).

A final comment on optimization is to simplify transfection conditions. Choosing a reporter that is easy to assay allows for testing a range of conditions quickly with minimal variability or potential complications. Ninety-six (96)-well plates are recommended because multiple variables and replicates can be tested in a single experimental plate. Small volumes minimize the use of medium and compounds, and sensitive assays are available to detect single or multiple reporters and biological markers in a single well. Once conditions are optimized for a specific cell type, they can be scaled to other plate formats.


Figure 1. Transfection method influences the maximum achievable protein expression and viability. HEK293 cells were transiently transfected with psiCheck™-2 Vector. Lipid-based transfection reagents, A, B, and FuGENE HD, were optimized according to the manufacturers’ instructions. Cell viability (CellTiter-Fluor Viability Assay) and reporter activity (ONE-Glo Luciferase Assay System) were then assayed using the GloMAX®-Multi Detection System Instrument. Data are the average of replicate samples ± SEM.

FuGENE HD

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.


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.

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.


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.

Multiplexing to Improve Optimization

Tracking cell viability along with reporter activity is critical to determining optimal transfection conditions. High reporter activity may come at the expense of cell health, and unhealthy cells are less likely to exhibit consistent, physiologically relevant responses.

Optimization is simplified further by using reporter and viability assays that can be measured in the same sample. The ONE-Glo™ Luciferase Assay System and the CellTiter-Fluor™ Cell Viability Assay  are compatible assays. By multiplexing these two assays, both reporter activity and viability can be measured in the same well of a 96-well plate in less than an hour. No medium changes or washing are needed.


Summary

Empirically determining optimal transfection conditions for any given cell type allows for the best return from your cell-based experiments. Optimal conditions should yield maximum reporter activity with minimum impact on cell health, thus preserving the biology of the cells for subsequent manipulation. By understanding the keys to successfully transfecting plasmid DNA, following a standard optimization plate layout, and multiplexing reporter and viability assays, optimal parameters can be determined with relative ease.


























Trista Schagat is scientific applications manager, and Kevin Kopish (kevin.kopish@promega.com) is strategic marketing manager at Promega. An online resource for analysis of optimization results following the standard plate format used here is available at: www.promega.com/techserv/tools/FuGeneHD/optimization.htm.

All registered trademarks or trademarks are property of Promega except FuGENE, which is a trademark of Fugent.

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