Lipofectamine 2000

Invitrogen™ Lipofectamine™ 2000 Transfection Reagent is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) into eukaryotic cells providing the following advantages:

• Highest transfection efficiency in many cell types and formats (e.g. 96-well). Refer to the Cell Lines database for a list of cell types successfully transfected.
• Nucleic acid-Lipofectamine 2000 complexes can be added directly to cells in culture medium, in the presence or absence of serum.
• It is not necessary to remove complexes or change/add medium after transfection, but complexes may be removed after 4-6 hours.

Guidelines for transfection

Use the procedure on below to transfect cells with short interfering RNA (siRNA) or Invitrogen™ Stealth RNAi™.

• Use the procedure on below to transfect cells with plasmid DNA.
• We recommend Gibco™ Opti-MEM™ I Reduced Serum Medium (Cat. No. 31985-062) to dilute Lipofectamine 2000 and nucleic acids before complexing.
• Do not add antibiotics to media during transfection as this causes cell death.
• Maintain the same seeding conditions between experiments.
• Test serum-free media for compatibility with Lipofectamine 2000 since some serum-free formulations (e.g. CD293, SFM II, VP-SFM) may inhibit cationic lipid-mediated transfection.

Note: For more tips for your RNAi experiment, refer to “Seven Steps to RNAi Success”. This manual is available from www.lifetechnologies.com/RNAi or Technical Service, as are cell-type specific RNAi transfection protocols (see “RNAi protocols”.)

Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, Sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of CHO-K1 cells with a reporter plasmid.

Use this brief procedure to transfect Stealth RNAi or siRNA into mammalian cells in a 24-well format. For other formats, see Scaling Up or Down Transfections. All amounts and volumes are given on a per well basis. Use this procedure as a starting point; optimize transfections as described in "Optimizing Stealth RNAi or siRNA transfection", especially if you are transfecting a mammalian cell line for the first time.

1. One day before transfection, plate cells in 500 μl of growth medium without antibiotics such that they will be 30-50% confluent at the time of transfection. Note: Transfecting cells at a lower density allows a longer interval between transfection and assay time, and minimizes the loss of cell viability due to cell overgrowth.


2. For each transfection sample, prepare oligomer-Lipofectamine 2000 complexes as follows:
   • a. Dilute 20 pmol Stealth RNAi or siRNA oligomer in 50 μl Opti-MEM I Reduced Serum Medium without serum (final concentration of RNA when added to the cells is 33 nM). Mix gently.
   
   
   • b. Mix Lipofectamine 2000 gently before use, then dilute 1 μl in 50 μl Opti- MEM I Reduced Serum Medium. Mix gently and incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.
   
   
   • c. After the 5-minute incubation, combine the diluted oligomer with the diluted Lipofectamine 2000. Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy).



3. Add the oligomer-Lipofectamine 2000 complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. Incubate the cells at 37°C in a CO₂ incubator for 24-96 hours until you are ready to assay for gene knockdown. Medium may be changed after 4-6 hours.

To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying RNA and Lipofectamine 2000 concentrations. Test 10-50 pmol RNA and 0.5-1.5 μl Lipofectamine 2000 for 24- well format. Depending on the nature of the target gene, transfecting cells at higher densities may also be considered when optimizing conditions.

Use the following procedure to transfect DNA into mammalian cells in a 24-well format. For other formats, see "Scaling up or down transfections". All amounts and volumes are given on a per well basis. Prepare complexes using a DNA (μg) to Lipofectamine 2000 (μl) ratio of 1:2 to 1:3 for most cell lines. Transfect cells at high cell density for high efficiency, high expression levels, and to minimize cytotoxicity. Optimization may be necessary (see "Optimizing plasmid DNA transfection").

1. Adherent cells: One day before transfection, plate 0.5-2 x 10⁵ cells in 500 μl of growth medium without antibiotics so that cells will be 70-90% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 4-8 x 10⁵ cells in 500 μl of growth medium without antibiotics.


2. For each transfection sample, prepare complexes as follows:
   • a. Dilute DNA in 50 μl of Opti-MEM I Reduced Serum Medium without serum (or other medium without serum). Mix gently.
   
   
   • b. Mix Lipofectamine 2000 gently before use, then dilute the appropriate amount in 50 μl of Opti-MEM I Medium. Incubate for 5 minutes at room temperature. Note: Proceed to Step c within 25 minutes.
   
   
   • c. After the 5 minute incubation, combine the diluted DNA with diluted Lipofectamine 2000 (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.



3. Add the 100 μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.


4. Incubate cells at 37°C in a CO₂ incubator for 18-48 hours prior to testing for transgene expression. Medium may be changed after 4-6 hours.


5. For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add selective medium (if desired) the following day.

To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as DNA and Lipofectamine 2000 concentrations. Make sure that cells are greater than 90% confluent and vary DNA (μg): Lipofectamine 2000 (μl) ratios from 1:0.5 to 1:5.

To transfect cells in different tissue culture formats, vary the amounts of Lipofectamine 2000, nucleic acid, 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.
 
Note: You may perform rapid 96-well plate transfections by plating cells directly into the transfection mix. Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 µl volume. Cells will adhere as usual in the presence of complexes.

¹  Surface areas may vary depending on the manufacturer.
²  Volumes of dilution medium in Step 2a & 2b of DNA or RNAi transfection protocols.