Lipofectamine 2000 CD reagent is a proprietary animal origin-free formulation for the transfection of nucleic acids into eukaryotic cells. Using Lipofectamine 2000 CD reagent for transfection provides the following advantages:
- Highest transfection efficiency in many cell types and formats.
- The animal origin-free formulation helps ensure that mammalian cell culture and bioproduction processes are free of animal-derived materials.
- DNA-Lipofectamine 2000 CD reagent complexes can be added directly to cells in culture medium.
- It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.
Important guidelines for transfection
- Culture cells in serum-free media that are free of animal-derived components. Test serum-free media for compatibility with Lipofectamine 2000 CD reagent since some serum-free formulations (e.g. CD 293, 293 SFM II, CD Hybridoma) may inhibit cationic lipid-mediated transfection.
- For consistent animal origin-free transfection, use OptiPro SFM (Cat. No. 12309-019) to dilute DNA and Lipofectamine 2000 CD reagent before complexing.
- Transfect cells at high cell density: For adherent cells, 90–95% confluence at the time of transfection is recommended for high efficiency, high expression levels, and to minimize cytotoxicity. For suspension cultures, transfect cells at a density of 0.8-1.1 x 106 cells/mL. Optimization may be necessary. Maintain the same seeding conditions between experiments.
- Do not add antibiotics to media during transfection as this causes cell death.
- Do not add Pluronic reagent or charged media extracts (e.g. dextran sulfate) to media during transfection as these reagents can inhibit transfection
Use the following procedure to transfect mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections. Use the recommended Lipofectamine 2000 CD regent amount as a starting point, then optimize conditions for your cell line and serum-free medium, as needed. All amounts and volumes are given on a per well basis.
- One day before transfection, plate 0.5-2 x 105 cells in 500 μL of growth medium without antibiotics so that they will be 90–95% confluent at the time of transfection.
- For each transfection sample, prepare complexes as follows
- Dilute DNA in 50 μL of OptiPro SFM. Mix gently.
- Mix Lipofectamine 2000 CD reagent gently before use, then dilute the appropriate amount in 50 μl of OptiPro SFM. Incubate for 5 minutes at room temperature. Note: Combine diluted Lipofectamine 2000 CD reagent with diluted DNA within 30 minutes.
- After 5 minute incubation, combine the diluted DNA with diluted Lipofectamine 2000 CD reagent (total volume = 100 μL). Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy). Note: Complexes are stable for 6 hours at room temperature.
- Add the 100 μL of complexes to a well containing cells and medium. Mix gently by rocking the plate back and forth.
- Incubate cells at 37°C in a CO2 incubator for 18–48 hours prior to testing for transgene expression. It is not necessary to change the medium, but medium may be replaced after 4–6 hours.
- For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add selective medium the following day.
To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine 2000 CD reagent, DNA, cells, and medium used in proportion to the relative surface area, as shown in the table. With automated, high-throughput systems, a complexing volume of 50 μl is recommended for transfections in 96- well plates.
|Culture vessel||Surf. area|
|DNA (μg) in|
media vol. (μL)
2000 CD (μL) in
media vol. (μL)
|96-well||0.3||0.2||100 μL||0.2 μg in 25 μL||0.5 μl in 25 μL|
|24-well||2||1||500 μL||0.8 μg in 50 μL||2.0 μl in 50 μL|
|12-well||4||2||1 ml||1.6 μg in 100 μL||4.0 μl in 100 μL|
|35-mm||10||5||4.0 μg in 250 μL||10 μl in 250 μL|
|6-well||10||5||2 mL||4.0 μg in 250 μL||10 μl in 250 μL|
|60-mm||20||10||5 mL||8.0 μg in 0.5 mL||20 μl in 0.5 mL|
|10-cm||60||30||15 mL||24 μg in 1.5 mL||60 μl in 1.5 mL|
Note: Surface areas are determined from actual measurements of tissue culture vessels, and may vary depending on the manufacturer.
Use the following procedure to transfect suspension cells at any scale. Before beginning, make sure that cells are healthy and >90% viable.
- On the day of transfection, prepare a single cell suspension from stock cells at <3 x 106 cells/mL. Seed cells at a density of 0.8–1.1 x 106 cells/ml in growth media without antibiotics.
- For each transfection sample, prepare complexes as in Step 2, using the following reagent amounts and volumes for every ml of cells transfected:
- Dilute 0.5–1.5 μg DNA in 34 μL of OptiPro SFM
- Dilute 1–10 μl of Lipofectamine 2000 CD reagent in 34 μL of OptiPro SFM
- Add the complexes to the flask containing cells and media.
- Incubate the cells at 37°C in a CO2 incubator on an orbital shaker rotating at 125 rpm for 24–96 hours prior to testing for transgene expression.
To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying cell density and Lipofectamine 2000 CD reagent concentrations.
Lipofectamine 2000 CD reagent is tested for the absence of microbial contamination using blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.