Optimal DNA delivery into mammalian cells depends on many factors and is cell-specific. General or published conditions can be used as a guide, but for best results we recommend optimizing transfection conditions for all cultured cells. Here we use FuGENE® HD Transfection Reagent to illustrate the keys to successful transfections, as well as the variables that should be tested when first optimizing transfection conditions. Optimization is not just about getting your cells to take up as much DNA as possible, then express as much protein as possible. It is about finding the optimal balance between maximal protein expression and minimal impact on cell viability. Through optimization, you can gain a solid understanding of the ranges in which you can expect the best possible outcome from your transfected cells.
Keys to Success
A number of factors will contribute to transfection success as well as the biological response of your transfected cells. Consider each of the following carefully.
Cell health: Cells should be actively dividing, passaged regularly in fresh growth medium and not allowed to become overconfluent prior to or at the time of transfection. Ideally, cells will be 75–90% confluent and greater than 95% viable (e.g., by trypan blue exclusion) at the point of harvest for transfection plating, and typically 80% confluent on the day of transfection using the FuGENE® HD Transfection Reagent. Passage number should be monitored because the cell's biological responsiveness can be unreliable at very 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 unintended cellular responses such as cytotoxicity or proinflammatory cytokine production. Preparation of plasmid DNA using a method qualified to produce transfection-grade DNA (e.g., PureYield™ Plasmid Purification Systems) will help you avoid these issues.
Transfection method: Methods include calcium phosphate-, lipid-, and electricity-mediated approaches. Lipid-based reagents are most popular, tend to give the lowest toxicity and have been used to transfect a wide range of cell lines. These methods do not require specialized equipment, and newer reagents involve a single addition of DNA:lipid complexes to cells with no subsequent medium change. However, not all reagents work to the same degree (Figure 1). Even under optimal conditions, the maximum protein expression and cell viability achieved can vary greatly. The optimum transfection technology is one that yields the highest possible protein expression with little to no discernable effect on cell health.
Simplicity: When first optimizing transfection conditions, keep things simple. Choose a reporter that is easy to assay so that you can test a range of conditions quickly with minimal potential complications or variability due to complex assay methods. Once you determine the optimal conditions for your cell line of interest, these conditions can be applied to all of your transfections. If you use a different cell line, you will need to optimize again.
Another recommendation to keep it simple is the plate format. 96-well plates are routinely used because multiple variables and replicates can be tested in a single experiment in a single 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(1)
. Once conditions are optimized for your cell type, they can be scaled to other well or flask sizes as needed for larger scale protein production or imaging.
Optimize Transfections with FuGENE® HD Transfection Reagent
Optimal transfection conditions for your cell line should be determined empirically. These conditions will be the foundation for many future experiments and data, so it is worthwhile to spend time up front to learn how to get the most from your cells. Optimal conditions will be those conditions that give you the highest reporter activity with minimal impact on cell health.
The FuGENE® HD Transfection Reagent is lipid-based, simple-to-use and can yield high transfection efficiencies with minimal cyototoxicity. An example showing optimization of transfection conditions for the FuGENE® HD Reagent in a 96-well plate is shown in Figure 2. Test variables include the ratio of reagent to DNA and volume of transfection mix added. 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 cationic lipid of the reagent), and the volume of this mixture determines how much DNA is administered. More is not necessarily better; more may lead to reduced protein expression and negatively impact cell health (Figure 3). Typical ratios are between 1.5:1 and 4:1 with addition of 2–10µl per well. In this experiment, optimal conditions for HEK-293 transfection were 5µl of a 2.5:1 mix.
The optimization scheme and plate layout in Figure 2 can be applied to test additional optimization variables.
Incubation period of DNA and FuGENE® HD Reagent prior to addition to cells: The typical incubation time is 0–15 minutes. For HEK-293 cells, both 0 minutes and 30 minutes showed greater variability in cell viability and lower reporter activity (data not shown).
Cell density: Generally, we recommend 1–2 × 104 adherent cells per well or 2–10 × 105 suspension cells per well.
Time after transfection to assay activity: Typically, 24 to 48 hours between transfection and activity assay is sufficient. The optimal time period depends on the protein being expressed, and thus the sensitivity of the assay available to detect it and the elements in the vector regulating expression. This time should be optimized for each protein expressed and based on the specific experimental needs (e.g., minimum expression time or maximum expression possible).
Several controls must be included in the optimization experiment. Untransfected cells are used as an indication of maximum viability and no reporter expression. DNA- and FuGENE® HD-only controls are included to monitor any unexpected effects of the transfection mix components on the cells.
Multiplexing for Easy Optimization
Tracking cell viability along with reporter activity is critical to determine optimal transfection conditions. High reporter activity may come at the expense of cell health, and unhealthy cells are less likely to show consistent, physiologically relevant biological responses.
Keep optimization simple by using reporter and viability assays that can be measured in the same sample. The ONE-Glo™ Luciferase Assay System for measuring firefly luciferase (Cat.# E6110) and the
CellTiter-Fluor™ Cell Viability Assay (Cat.# G6080) are examples of compatible assays(3)
. 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 is needed.
Empirically determining optimal transfection conditions for your cell type will allow you to get the most out of your experiments. Optimal conditions will give you maximum reporter activity with minimum impact on cell health, thus preserving the biology of your cells for subsequent manipulation. By understanding the keys to success, following a standard optimization plate layout and multiplexing reporter and viability assays, optimal parameters can be determined with relative ease.
For rapid analysis of your optimization results following this standard plate format, use the FuGENE® HD Transfection Reagent Optimization Analysis Worksheet. For a list of conditions that have been used to transfect various cell types, visit the FuGENE® HD Transfection Reagent Protocol Database.