Transfecting Plasmid DNA into RAW264.7 Macrophage Cells Using Lipofectamine LTX Reagent

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®  LTXReagent.
• 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®  LTXReagent 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.

Materials Needed

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

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.

1. The day before transfection, trypsinize and count the cells. Plate 6.2X10⁴ cells per well in 0.5ml of complete growth medium.


2. (Optional) The day of transfection, remove growth medium from cells and replace with 0.5ml of complete growth medium.


3. 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.


4. 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.


5. 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.


6. 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.


7. 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.