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.