Efficient Transfection of Primary Neuronal Cells with TransFectin™ Lipid Reagent TransFectin lipid reagent demonstrates gentle yet effective transfection of primary rat hippocampal neurons.
Efficient tranfections of primary neuronal cells with TransFectin lipid reagent. Primary rat hippocampal neurons were transfected on coverslips in 24-well plates using 1 µg of a GFP expression plasmid and 2 µl TransFectin. At 3 hr after complex addtion, the medium was replaced with 50:50 conditioned:fresh medium. Neurons were fixed and analyzed 48 hr after transfection. Data courtesy of Dr. Janil Kanaani, Diabetes Center, University of California, San Francisco.
Protocol for Transfection of Rat Hippocampal Neurons with TransFectin Lipid Reagent
Primary hippocampal neurons were prepared from embryonic rat brains (E18/E19) and were seeded on glass coverslips coated with poly-D-lysine in 24-well plates.
The cells were cultured at a density of approximately 7.5 x 104 cells/well in 0.5 ml of neurobasal medium with B27 supplement, 0.5 mM glutaMAX, and penicillin/streptomycin.
Cells were transfected at day in vitro 6 (DIV 6) using either 1:2 or 1:4 ratio between DNA and TransFectin in neurobasal medium (1 µg DNA and 2 µl or 4 µl TransFectin per well).
After 3 hr incubation at 37°C in a 5% CO2 incubator, the transfection medium was replaced with 50:50 conditioned:fresh medium.
Neurons were fixed 48 hr posttransfection with 4% paraformaldehyde.
Constructs used for transfection are: (1) pEGFP-N3 (4.7 kb) from CLONTECH; (2) fusion protein of Glutamic acid decarboxylase 65 and GFP (GAD65-GFP; 6.4 kb). Data courtesy of Dr Jamil Kanaani, Diabetes Center, University of California, San Francisco.
Effective Delivery into CHO and NIH3T3 Cells Using TransFectin Lipid Reagent TransFectin lipid reagent demonstrates high transfection efficiency of CHO and NIH3T3 cells.
CHO and NIH3T3 cultures were transfected with pCMV.SPORT-β-gal using Bio-Rad's TransFectin lipid reagent. Cells were stained with X-gal to determine β-galactosidase activity 24 hr posttransfection. Blue cells indicate successful transfection.
Lower Cytotoxicity: Increased Viability TransFectin™ lipid reagent provides exceptional performance with less cytotoxicity than is typically observed in other high-efficiency transfection reagents. Very high efficiencies may be obtained while maintaining the viability of your cells. The combination of high efficiency and lower cytotoxicity results in greater levels of reporter gene expression.
Total protein assays provide an accurate assessment of culture health after transfection. Bradford assays performed on cell cultures 24 hr posttransfection indicate that TransFectin-treated cultures are typically healthier than cultures exposed to other lipid reagents.
Cytotoxicity comparisons based on total protein assays.A, COS-7 and B, HeLa cells were transfected at 90% confluence using TransFectin (orange) and another cationic lipid reagent (blue) in a single 24-well culture vessel. Total protein was determined using the Bradford reagent 24 hr posttransfection. Higher OD values correlate with more protein, allowing an assessment of the relative toxicity of the lipids.
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.
High Performance at Multiple Confluences The TransFectin™ lipid reagent protocol allows you to achieve high efficiencies across a wide range of culture confluences, while maintaining a high level of consistency and performance. Perform transfections with or without serum-containing medium at a culture confluences between 40% and 90%.
COS-7 (orange) and HeLa (blue) cells were transfected at confluence between 40% and 90% with pCMV.SPORT-β-gal. Cell cultures were assayed using ONPG 24 hr posttransfection to quantitate expression.
Consistency Using a Variety of Vessel Sizes Achieve consistent levels of expression with a variety of cell culture vessels. The graph below shows the amount of expression achieved using different-sized culture vessels.
A comparison of β-galactosidase expression in different-sized culture vessels. HeLa cells were transfected with pCMV.SPORT-β-gal at 70% confluences, in the presence of serum, using TransFectin lipid reagent. Cells were assayed for β-galactosidase activity using ONPG 24 hr posttransfection to quantitate expression. Results show that reporter gene activity is scalable with vessel size when using TransFectin lipid reagent.
Suggested Reagent Quantities for Different Sizes of Plates/Wells
The table below provides approximate reagent requirements for the most popular vessels. For unlisted vessels, simply determine the change in surface area and linearly scale the reagent volume.
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)
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.
For each well, prepare plasmid in 50 µl serum-free medium. Use 0.25 to 1.0 µg plasmid for each 50 µl medium.
Prepare TransFectin in 50 µl serum-free medium for each well. Use 0.25 to 4.0 µl lipid for each 50 µl medium.
Mix the plasmid and TransFectin solutions together. Gently mix by tapping or pipetting. Incubate 20 min at room temperature.
Add the 100 µl DNA-TransFectin complexes to the cell suspension; rock the plate gently to ensure adequate mixing of the solutions.
Incubate the cells at 37°C in a 5% CO2 incubator. Additional medium may be added 4 to 6 hr after addition of complexes.
For transient expression, assay reporter gene activity 24–48 hr after transfection.
Protocol for Transfection of Adherent Cells (24-Well Plates)
The day before transfection, inoculate 24-well plates with an appropriate number of cells in serum-containing medium so that they will be 50 to 90% confluent the following day. For most cell lines, plating 0.5 to 8.0 x 105 cells in 0.5 ml medium should be appropriate. Incubate the cells at 37°C in a 5% CO2 incubator overnight.
For each well, prepare plasmid in 50 µl serum-free medium. Use 0.25 to 1.0 µg plasmid DNA for each 50 µl serum-free medium.
Prepare the TransFectin™ lipid reagent in 50 µl serum-free medium. Use 0.25 to 4.0 µl for each well as a starting point for optimizing the reaction.
Mix the DNA and TransFectin solutions together. Gently mix by tapping or pipetting. Incubate 20 minutes at room temperature.
Add 100 µl of the DNA:TransFectin complexes directly to cells in serum-containing medium. Swirl gently. Incubate the cells at 37°C in a 5% CO2 incubator. Additional medium may be added 4 to 6 hr after addition of complexes.
For transient expression, assay for reporter gene activity 24 to 48 hr posttransfection.
For stable expression of the transfected plasmid sequence, remove transfection medium 24 hr after transfection and trypsinize the cells. Transfer the cells to a fresh plate with growth medium containing no selective agent. The following day, replace with new medium containing the selective agent. Continue incubating for 1 to 2 weeks to allow growth of the cells expressing the transgene.
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
Culture Vessel Size
Surface Area (cm2)
Volume of Plating Media
Plasmid DNA
Volume of Serum Free Medium
TransFectin Reagent
96-well
0.32
0.1 ml
50–200 ng
20–25 µl
0.1–0.6 µl
24-well
1.9
0.5 ml
0.25–1.0 µg
50–100 µl
0.25–4.0 µl
12-well
3.8
1.0 ml
0.50–2.0 µg
100–200 µl
1.0–8.0 µl
6-well/35 mm
9.2
2.0 ml
1.0–4.0 µg
250–500 µl
2.5–15 µl
60 mm
21.0
5.0 ml
2.0–8.0 µg
500–1000 µl
5–20 µl
100 mm
60.0
10.0 ml
12–36 µg
1.5–3.0 ml
15–60 µl
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
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).
The Transfection Protocol Online Library contains protocols obtained from the literature, developed by Bio-Rad scientists, or submitted by scientists like you. browse protocols to view our library and find your starting point.
Bio-Rad Laboratories has helped scientists deliver molecules into cells for over 35 years. Instruments, including the Gene Pulser® series of electroporators, the MicroPulser™ electroporator, and the Helios® gene gun and PDS-1000/He™ biolistic systems, have been used successfully to transfect prokaryotic and eukaryotic cells, including plant and animal cells. These transfection instruments are cited extensively in the literature. Our chemically mediated methods include lipid-based transfection reagents — TransFectin™ lipid reagent for delivery of plasmid DNA and siLentFect™ lipid reagent for siRNA delivery.
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Optimization of TransFectin Lipid Reagent-Mediated Transfection for Different Cell Types
