Lipofectamine LTX and PLUS Reagents

Description

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 our website 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.lifetechnologies.com/transfection for cell-specific transfection protocols.

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.

1. 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.


2. 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.


3. Add ~100 μl DNA-lipid complex (step 2 d, above) dropwise to the well containing cells. Mix gently by rocking the plate back and forth.


4. Incubate the 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.

Use these guidelines for high-throughput transfections, or if dispensing small amounts of reagents. Pre-dilute the reagents first, and then add this larger volume 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.

1. 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


2. 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.



3. Add ~20 μl DNA-Lipofectamine® LTX complexes to each well containing cells. Mix gently by rocking the plate back and forth.


4. Incubate at 37°C in a CO₂ incubator for 18–48 hours and test for transgene expression. Medium may be changed after 4–6 hours.

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.

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.

This product is covered by Limited Use Label License No.5: Invitrogen Technology (see the catalog or our website). By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses.