1. The plasmid vectors encoding the fusion proteins are mixed in sterile electroporation cuvettes and empty vector DNA is used to keep the total amount of DNA constant for a given experiment. The amount of DNA per cuvette typically ranges from 5 to 30 ug and optimal concentrations must be determined by experimentation. Both 0.2 or 0.4 cm gap cuvettes are available, and electroporation conditions must be optimized for each cell line.

2. Rinse the cell monolayer with phosphate buffered saline, and then briefly treat the cells with trypsin (0.05%) in 0.53 mM EDTA. Remove the trypsin-EDTA solution.

3. When cells begin to release from the surface of the flask, recover the cells in culture medium containing serum. Wash the cells 2 times by centrifugation in Dulbecco’s calcium-magnesium free phosphate buffered saline.

4. Resuspend the cells to a final concentration of approximately 1x10⁷ cells per ml in Dulbecco’s calcium-magnesium free phosphate buffered saline. Add 400 ul of the cell suspension to each 0.2 cm gap electroporation cuvette containing the DNA.

5. Gently mix the contents of the cuvette and then pulse the cells at the desired voltage and capacitance. Optimal electroporation conditions must be determined empirically for each different cell-type used. Voltage-capacitance curves should be generated using an easily measured reporter gene, such as CMV-luciferase. Electroporation conditions for some cell lines we use are provided below as reference for starting points to empirically determine the conditions that are suitable for a particular cell line and culture conditions.

The following conditions were determined using the BTX electroporator at a maximum voltage setting of 500 V, resistance setting R3 (48 ohms) using 0.2 cm gap cuvettes with 400 ul cell suspension in calcium-magnesium-free phosphate buffered saline. The typical pulse duration obtained under these conditions were 9 - 10 msec.

6. The cells are then immediately recovered from the cuvette and diluted in phenol red free tissue culture medium containing serum.

7. The cells are inoculated drop-wise onto a sterile 25 mm cover glass in 35 mm culture dishes or 42 mm cover glass in 60 mm culture dishes. The cells are allowed to attach to the cover glass for approximately 20 minutes prior to gently flooding the culture dish with media.

8. The cultures are then placed in an incubator for 18 h prior to imaging.

Department of Medicine, PO Box 800578, University of Virginia Health System, Charlottesville, VA 22908-0578