Lipofectamine® LTX Reagent is a proprietary, animal-origin free formulation for the transfection of DNA into eukaryotic cells with low cytotoxicity. This reference provides a recommended procedure to transfect plasmid DNA into RAW264.7 macrophage cells using Lipofectamine® LTX Reagent (Cat. No. 15338-100).
Important Guidelines for Transfection
Follow these important guidelines when transfecting mNPC cells using Lipofectamine® LTX Reagent:
- Maintain the same seeding conditions between experiments. Use low-passage cells; make sure cells are
healthy and greater than 90% viable before transfection.
- Transfection can be performed both in the presence or absence of serum. Test serum-free media for compatibility with Lipofectamine® LTX Reagent.
- Using PLUS Reagent (Cat. No. 11514-015) enhances transfection performance in RAW264.7
- We recommend Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute the DNA and Lipofectamine® LTX Reagent before complexing.
- Visit www.lifetechnologies.com/transfection or contact Technical Service for other specialized transfection protocols.
- Lipofectamine® LTX Reagent performs well with vector-based RNAi experiments. For siRNA and Stealth™ RNAi transfections, we recommend Lipofectamine® RNAiMAX (Cat. No. 13778-075). Go to www.lifetechnologies.com/RNAi or contact Technical Service for more information.
Have the following reagents on hand before beginning:
- RAW264.7 Macrophage Cells
- Plasmid DNA of interest (100 ng/μl or higher)
- Lipofectamine® LTX Reagent (store at +4°C until use), and PLUS™ Reagent (store at 4°C)
- Opti-MEM® I Reduced Serum Medium
- Appropriate tissue culture plates and supplies
Transfection of RAW264.7 Macrophage Cells
Use this procedure to transfect plasmid DNA into RAW264.7 Macrophage Cells s in a 48-well format. All amounts and volumes are given on a per well basis.
- The day before transfection, trypsinize and count the cells. Plate 6.2X104 cells per well in 0.5ml of complete growth medium.
- (Optional) The day of transfection, remove growth medium from cells and replace with 0.5ml of complete growth medium.
- For each well of cells to be transfected, dilute 0.3 μg of DNA into 40 μl of Opti-MEM® I Reduced Serum Medium without serum.
- Mix PLUS™ Reagent gently before use, then add 0.375 μl PLUS™ Reagent directly to the diluted DNA. Mix gently and incubate for 5-15 minutes at room temperature.
- For each well of cells, add 0.75-1.50 μl of Lipofectamine® LTX Reagent into the above diluted DNA solution, mix gently and incubate for 30 minutes at room temperature to form DNA-Lipofectamine® LTX complexes.
- After the 30 minute incubation, add 40 μl of the DNA-Lipofectamine® LTX complexes directly to each well containing cells and mix gently by rocking the plate back and forth.
- Complexes do not have to be removed following transfection. Incubate the cells at 37°C in a CO2 incubator for 18-24 hours post-transfection before assaying for transgene expression.