TransFectin Lipid Reagent

TransFectin™ Lipid Reagent Outperforms in Transfection Efficiency

TransFectin lipid reagent illustrates high transfection efficiency in a broad range of commonly used mammalian cell lines, as it outperforms competing lipids in the data shown. For some cell lines, transfection efficiencies >90% are routinely achieved using TransFectin. In addition, use less reagent per experiment than is needed for many other products, while still achieving excellent performance.

Relative activity in three cell lines. Cells were treated with pCMV.SPORT-β-gal using TransFectin lipid reagent (orange) and three other lipid reagents. Cell cultures were assayed using ONPG 24 hr posttransfection and OD readings were taken to quantitate β-galactosidase expression. The cells transfected with TransFectin clearly illustrate higher transgene activity, thus indicating greater expression and transfection efficiency.

Transfection efficiency comparisons. A variety of cell lines were transfected in 24-well plates with pCMV.SPORT-β-gal using Bio-Rad's TransFectin (orange) or another popular lipid transfection reagent (blue). At 24 hr posttransfection, cells were lysed and stained with X-gal. Efficiency is reported as the percentage of cells transfected (expressing β-galactosidase) in the population.

Effective delivery into CHO cells using TransFectin lipid reagent. A, B, C, CHO cultures after transfection using three commercially available lipid transfection reagents and D, TransFectin. Cells were stained with X-gal to determine β-galactosidase activity 24 hr posttransfection. Blue cells indicate successful transfection.

Effective Delivery into Suspension Cells

TransFectin™ lipid reagent illustrates efficient delivery into K562 (suspension) cells.

Efficient delivery. K562 cells were plated in 24-well plates at a density of 4 x 10⁵ cells/well. TransFectin: plasmid DNA complexes were formed by combining 1.25 µg (dark orange) or 1.00 ug (light orange) pCMV.SPORT-β-gal with TransFectin lipid reagent. After 20 min, transfection complexes were added to the cells in the presence of serum. At ~24 hr posttransfection, cells were pelleted and lysed, then assayed for β-gal expression.

Protocol for Transfection of Suspension Cells
(24-Well Plates)

1. The day before transfection, dilute the cells so that they will be in log phase growth the following day. For most cell lines, inoculating 0.75 to 8.0 x 10⁵ cells per 0.5 ml medium should be appropriate. Incubate the cells overnight at 37°C in a 5% CO₂ incubator.
2. For each well, prepare plasmid in 50 µl serum-free medium. Use 0.25 to 1.0 µg plasmid for each 50 µl medium.
3. Prepare TransFectin in 50 µl serum-free medium for each well. Use 0.25 to 4.0 µl lipid for each 50 µl medium.
4. Mix the plasmid and TransFectin solutions together. Gently mix by tapping or pipetting. Incubate 20 min at room temperature.
5. Add the 100 µl DNA-TransFectin complexes to the cell suspension; rock the plate gently to ensure adequate mixing of the solutions.
6. Incubate the cells at 37°C in a 5% CO₂ incubator. Additional medium may be added 4 to 6 hr after addition of complexes.
7. For transient expression, assay reporter gene activity 24–48 hr after transfection.

Optimization
Determining the optimum conditions for transfection efficiency is essential for maximizing gene expression and minimizing cellular toxicity. The two most important parameters to optimize transfection efficiency using TransFectin are: amount of TransFectin and concentration of nucleic acid. In general, gene expression will increase, plateau, and then decrease with increasing volumes of TransFectin. This decrease in gene expression correlates with reduced cell viability. As increasing amounts of plasmid are added to the cells, gene expression will increase, then plateau, or even decrease since the amount of DNA used can directly affect toxicity. If toxicity is encountered, try reducing lipid or DNA amounts used in the transfection. The concentration of TransFectin and the amount of plasmid required for maximal expression may vary from one cell line to another. The highest levels of expression can be obtained using amounts of TransFectin and DNA suggested in the table below for given culture vessel sizes.

Suggested Reagent Quantities for Different Sizes of Plates/Wells

Other Recommendations for Best Results

• For most cell lines use a ratio of DNA (µg) to lipid (µl) of 1:2–1:3 as a starting point to optimize conditions
• Invert the tube to mix contents before using
• TransFectin is designed to work in media containing serum but can be used in the absence of serum
• Use sterile polystyrene plastic ware (for example,12 x 75 mm tubes or multi-well trays) to prepare the plasmid solutions and lipid solutions. Polystyrene is recommended because cationic lipid-plasmid complexes may bind to polypropylene

Additional Optimization Resources

Optimization of TransFectin Lipid Reagent-Mediated Transfection for Different Cell Types

It is important to optimize transfection conditions not only for each transfection method but also for every cell type. Cell density, duration of transfection, volume of medium during transfection, and ratio of lipid reagent to DNA are key factors for efficiency. In this article we describe the optimization of transfection with TransFectin monitored by a secreted alkaline phosphatase (SEAP) reporter gene assay. We compare two suspension cell types that are considered difficult to transect: bovine peripheral blood mononuclear cells (PBMCs) and bovine B-lymposarcoma BL3.1 cells (Anderson et al. 2004, Fenton et al. 1998). Additionally, we optimize the transfection efficiency for two adherent cell types: primary bovine oviductal cells and a calf pulmonary artery endothelial cell line (CPAE).