The Eppendorf (www.eppendorf.com) Multiporator®, together with the specially designed Hypoosmolar Electroporation Buffer, creates a system for effectively transfecting eukaryotic cells without severely damaging them. The application of extremely short pulses, in the microsecond range (soft pulses), ensures high survival rates of the cells.
Additionally, the pulses are electronically regulated, which makes the parameters independent from the resistance properties of the sample and allows uniform and reproducible pulses.
The low-conductive buffer system markedly reduces the current flow, and the electrically induced pores are much larger than those obtained from pulses in conductive solutions. The components of the media are adapted to the cytosolic ion composition of the cell, which prevents the cellular Na+/K+ gradient from collapsing during electroporation. The low osmolarity of the hypoosmolar buffer lets the cells swell up and round off, thus enabling an easier and more controlled electroporation.
Crucial parameters for successful electroporation are the voltage, the length of the pulse (time constant, t), and the number of pulses used. The voltage, which has to be set on the device, depends on the cell type (cell diameter), gap width of the cuvette, and the temperature. It is advisable to carry out a series of experiments employing different values. Ideal pulse lengths for electroporation have proven to be 40–100 µs at room temperature and 15–40 µs at 4°C.
For most cell lines, electroporation is carried out with one pulse. If one pulse proves to be insufficient, two or more pulses may be used. During multiple pulsing operations, the Multiporator automatically maintains a 60-second interval between pulses to allow the cell membrane to regenerate.