Methods Transfection

1. Twenty-four hours before transfection, cells are split into six-well tissue culture plates and should be approx 50-80% confluent for the transfection procedure.

2. Prepare the following in sterile tubes: For each well of a six-well plate, dilute 2 ig of DNA and 5 iL of lipofectamine reagent into 1 mL serum-free medium (e.g., Optimem I Reduced Serum Medium), and vortex to mix (see Notes 1 and 2).

3. Incubate the solution at room temperature for 30 min to allow DNA-liposome complexes to form.

4. While the solution is incubating, wash the cells 1X in PBS, add 1 mL of Optimem I to the cells, and incubate at 37°C.

5. After the DNA-liposome complexes have been incubated for 30 min, remove the Optimem I medium from the cells and add 1 mL of the DNA-liposome solution to the cells.

6. Incubate the cells for 5 h at 37°C (see Note 3).

7. Following incubation, add 1 mL of complete growth medium containing twice the normal concentration of serum without removing the transfection mixture.

8. Replace the medium with fresh complete medium 18-24 h following the start of the transfection.

9. If cells are to be stimulated, they should be stimulated 48 h posttransfection, prior to harvesting cells (see Note 4).

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