Transfection of PC12 Cells with Lipofectamine

Cells

1. Cells should be plated on collagen-coated dishes, as for normal propagation. If there appears to be large-scale loss of cells after Lipofectamine treatment, then it might pay to try using polylysine-coated plates.

2. Plate 2X 10⁶ cells per 60 mm tissue culture dish in DMEM/10% horse serum/5% fetal calf serum/pen-strep (normal PC12 growth medium) and grow overnight in 10% CO₂ incubator. The cells should be 50–70% confluent.

3. Remove medium from cells, rinse 1X with 3 ml pre-warmed OptiMEM serum free medium (Gibco) and add 5 ml OptiMEM and put the cells back in the incubator until ready to transfect.

Transfection

1. For each dish of cells aliquot 2 micrograms of the plasmid or plasmid mixture into a 5 ml sterile tube. Add 100 microliters of OptiMEM medium.

2. In a separate tube aliquot 15 microliters of 2 mg/ml Lipofectamine (Gibco) and add 100 microliters OptiMEM.

3. Combine the diluted DNA and diluted Lipofectamine, mix gently, and let incubate at room temperature for 15–40 min. This allows the DNA and the Lipofectamine to form the complexes that will get the DNA into the cells.

4. To each transfection mixture add 2.8 ml pre-warmed OptiMEM.

5. Aspirate the medium from the dish of cells, add the diluted transformation mix, and incubate the cells with the mix in a 10% CO₂ incubator for approximately 5 h.

6. At the end of the incubation, remove the transformation mixture and add 5 ml of pre-warmed normal PC12 growth medium with serum. Return the cells to the 10% CO₂ incubator.

This protocol typical has given 1–2% transfected cells, as judged by staining with X-gal for a CMV-beta-galactosidase reporter gene. I usually let the cells grow for 40 to 60 h before harvesting in transient transfections.