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Optimize Transfection of Cultured Cells

Trista Schagat and Kevin Kopish

Promega Corporation


Protein expression from plasmid DNA transfected into cultured cells is a powerful tool to study protein function and monitor intracellular events. Optimal protein expression and cell viability require optimizing transfection conditions. We discuss how to optimize transfection conditions using FuGENE® HD Transfection Reagent and the factors that are most likely to influence success.


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.

Transfection method influences the maximum achievable protein expression and viability.
Figure 1. Transfection method influences the maximum achievable protein expression and viability. HEK293 cells were transiently transfected with psiCHECK™-2 Vector (Cat.# C8021), which expresses luciferase driven by the constitutive SV40 promoter. Three different lipid-based transfection reagents (A, B, and FuGENE® HD) were optimized according to the manufacturers’ instructions. Cell viability (as measured using the CellTiter-Fluor™ Viability Assay; Cat.# G6080) and reporter activity (as measured using the ONE-Glo™ Luciferase Assay System; Cat.# E6110) then were assayed using the GloMax®-Multi Detection System Instrument (Cat.# E7031). Data were generated using optimal conditions for each reagent(conditions that gave the highest reporter activity with relatively minor loss in viability). Data are the average of replicate samples ± SEM. For each optimization, a minimum of 3 lipid reagent:DNA ratios and 2 cell densities were tested.

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 cytotoxicity. 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.

FuGENE HD transfection optimization experiment.
Figure 2. FuGENE® HD transfection optimization experiment. Panel A. 96-well plate layout to determine optimal transfection conditions with the FuGENE® HD Transfection Reagent. DNA, FuGENE® HD and medium were used to prepare 100µl transfection mixes at the indicated reagent:DNA ratios in a separate 96-well plate. After a 15-minute incubation at room temperature, a multichannel pipette was used to add the transfection mix to each well of the transfection plate. The outer wells of the plate contained 100µl/well of medium only. All other wells contained 100µl/well of cells in growth medium. Column 2: Control cells were exposed to: 10µl of medium, 10µl of DNA (20ng/µl in medium), or 10µl of FuGENE® HD Reagent (from a mix of 100µl of medium + 8µl of FuGENE® HD Reagent). Columns 3–11: The indicated volume of transfection mix with the indicated ratio of FuGENE® HD Reagent to DNA was added to the cells. Panel B. Schematic of the optimization experiment. Day 1, plate is seeded with 100µl of medium or healthy cells (harvested from a ~75–90% confluent flask). Day 2, the appropriate controls are assembled and DNA:FuGENE® HD Reagent transfection mixes are added to cells as indicated in Panel A. Day 3 or 4, cell viability and reporter activity are assayed.
HEK-293 transfection optimization experiment.
Figure 3. HEK-293 transfection optimization experiment. Optimal transfection conditions for HEK-293 with the  FuGENE® HD Reagent were determined using the protocol recommended in Figure 2. HEK-293 cells were grown to 85% confluency, then harvested and plated in a 96-well plate at 2 × 104 cells/100µl/well. The next day, the pGL4.13 Vector (Cat.# E6681) expressing firefly luciferase under a constitutive SV40 promoter was diluted in 100µl of serum-free medium (DMEM) to 20ng/µl and mixed with 3–8µl of FuGENE® HD Reagent to achieve the indicated reagent:DNA ratio. After a 15-minute incubation, 2µl, 5µl or 10µl was added per well. The cells were mixed gently, then incubated for 24 hours at 37°C, 5% CO2. Cell viability (as measured by the CellTiter-Fluor™ Viability Assay) and reporter activity (as measured by the ONE-Glo™ Luciferase Assay System) then were assayed using the GloMax®-Multi Detection System. For each optimization, a minimum of three lipid:DNA rations and two cell densities were tested. Data are the average of replicate samples ± SEM.

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. 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.


  1. Fan, F. and Wood, K. (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 5, 127–36.

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