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.