Related Product Information
Lipofectamine® LTX Reagent is a proprietary, animal-origin free cationic lipid formulation for the transfection of DNA into eukaryotic cells that offers:
- Highest transfection expression performance with low cytotoxicity in many cell types and formats (e.g. 96-well).
- Easy protocol for DNA-Lipofectamine® LTX complex formation.
- Reduced cytotoxicity of Lipofectamine® LTX Reagent allows the use of a greater range of lipid doses, enabling excellent transfection results despite differences in cell density, minor pipetting inaccuracies, and other variations.
- For many cell lines, using PLUS™ Reagent (also available separately, Cat. no. 11514-015) enhances transfection performance.
Guidelines for Transfection
- For best results, optimize DNA and lipid amounts as described in Optimizing Transfections before following the Standard Protocol or the High-Throughput Protocol.
- The addition of antibiotics to media during transfection may result in cell death in some cell lines. Test each cell line individually.
- Lipofectamine® LTX Reagent performs well with vector-based RNAi experiments. For siRNA and Stealth™ RNA transfections, we recommend Lipofectamine® RNAiMAX (Cat. no. 13778-075).
- Visit www.invitrogen.com/transfection or contact Technical Support for specialized transfection protocols (including cell-type specific advice on use of PLUS™ Reagent and antibiotics, and a protocol for vector-based RNAi).
- We recommend Opti-MEM® I Reduced Serum Medium (Cat. no. 31985-062) to dilute the DNA and Lipofectamine® LTX Reagent before complexing.
- Maintain the same seeding conditions between experiments. Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine® LTX Reagent.
To obtain the highest transfection performance, perform optimization by using a range of DNA and Lipofectamine® LTX amounts with and without the PLUS™ Reagent as described below for a 24-well format. For other formats, adjust the amounts according to Scaling Up or Down Transfections (below). Consider varying cell density for further optimization. Note: Visit www.invitrogen.com/transfection for cell-specific transfection protocols.
|Cells||DNA||Lipofectamine® LTX||PLUS™ Reagent|
|Sensitive cells (HeLa, HT1080)||250 ng||0.375 μl – 1.25 μl||0.125 μl – 0.5 μl|
|Most cell lines||500 ng||0.75 μl – 3.0 μl||0.25 μl – 1.0 μl|
|750 ngl||1.125 μl – 4.5 μl||0.375 μl – 1.5 μl|
|Suspension and robust cells1||1000 ng||1.5 μl – 5.0 μl||0.5 μl – 2.0 μl|
1 Examples are MCF7, A549, Jurkat, THP1 and HL60
Use these guidelines to transfect DNA into mammalian cells in a 24-well format after optimizing the reactions as described above. If using the PLUS™ Reagent, start with a 1:1 ratio of DNA (μg) to PLUS™ Reagent (μl). All amounts are given on a per well basis. Note: Read Important Guidelines for Transfection (above) before using antibiotics.
- Adherent cells: One day before transfection, plate cells in 500 μl of growth medium so that the cells will be 50–80% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 100,000–250,000 cells in 500 μl of growth medium.
- For each transfection sample, prepare complexes as follows:
- a. Dilute the optimized amount of plasmid DNA in 100 μl Opti-MEM® I Reduced Serum Medium. Mix thoroughly.
- b. Only if using PLUS™ Reagent: Mix PLUS™ Reagent gently before use, add the optimized volume of PLUS™ Reagent directly to the diluted DNA. Mix gently and incubate for 5 minutes at room temperature.
- c. Mix Lipofectamine® LTX gently before use, and add the optimized volume directly to the diluted DNA. Mix thoroughly.
- d. Incubate for 30 minutes at room temperature. DNA-lipid complexes are stable for 6 hours at room temperature.
Use these guidelines for high-throughput transfections, or if dispensing small amounts of reagents. Pre-dilute th reagents first, and then add this largerAvolume to the diluted DNA. Discard unused diluted reagents, as diluted lipid loses activity after 5 minutes. All amounts are given on a per well basis for the 96-well format; for other formats, refer to Scaling Up or Down Transfections (below). For best results, optimize transfections as described in Optimizing Transfections
Note: Review Important Guidelines for Transfection (above) before using antibiotics.
- Adherent cells: One day before transfection, plate cells in 100 μl of growth medium so that the cells will be 50–80% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes, plate 20,000–50,000 cells in 100 μl of growth medium without antibiotics
- For each transfection sample, prepare DNA-lipid complexes as follows:
- Dilute the optimized amount of plasmid DNA in 10 μl Opti-MEM® IReduced Serum Medium. Mix gently.
- Only if using PLUS™ Reagent: Mix PLUS™ Reagent gently. Prepare a 1:10 dilution of PLUS™ Reagent in Opti-MEM® I Reduced Serum Medium. Add the optimized volume of diluted PLUS™ Reagent to diluted DNA. Mix gently and incubate 5 minutes at room temperature.
- Mix Lipofectamine® LTX gently. Prepare a “Master Mix” by making a 1:10 dilution of Lipofectamine® LTX in Opti-MEM® I Reduced Serum Medium. Aliquot the appropriate amount of the “Master Mix” (equivalent to the optimized amount of Lipofectamine® LTX), and bring up the final volume to 10 μl per well with Opti-MEM® I Reduced Serum Medium. Mix gently.
- Add the diluted Lipofectamine® LTX to the diluted DNA. Mix gently. Proceed to the next step within 5 minutes
- Incubate for 30 minutes at room temperature. DNA-lipid complexes are stable for 6 hours at room temperature.
To transfect cells in different tissue culture formats, vary the amounts of DNA, Lipofectamine® LTX, PLUS™ Reagent cells, and medium used in proportion to the relative surface area, as shown in the table below and given on a per well basis. Determine the optimal amount required for Lipofectamine® LTX, plasmid DNA, and PLUS™ Reagent (if used) as described in Optimizing Transfections for 24-well format.
|Culture vessel||Surface area per well 1||Volume plating medium||Volume dilution medium 2||Relative Surface Area (compared to 24-well)|
|96-well||0.3 cm2||100 μl||20 μl||0.2X|
|48-well||1.0 cm2||200 μl||40 μl||0.4X|
|24-well||2 cm2||500 μl||100 μl||1X|
|12-well||4 cm2||1 ml||200 μl||2X|
|6-well||10 cm2||2 ml||500 μl||5X|
1 Surface areas may vary depending on the manufacturer.
2 Lipofectamine® LTX and DNA are diluted separately in the High Throughput Protocol, each into one half of the total volume of dilution medium.