Design Your Own Electroporation Protocol Episode 4 - 

Temperature Considerations

By Michelle M. Ng, Ph. D.


Optimizing Electroporation

This is the fourth in a series of posts where we will be providing tips for developing or improving your own electroporation method.

The majority of mammalian cells are electroporated efficiently at room temperature.  However there are couple reasons to consider an electroporation experiment at a lower temperature such as 4  ͦC:

  • To reduce cell heating. As mentioned earlier in Episode 3 as you increase volume you may also increase conductance and heating of the sample which can have negative effects on cell viability. Electroporation programs with high voltage pulses (typically with bacteria), long pulse durations or multiple pulses may also cause heating of the cells
  • To keep pores open longer after electroporation. Since electroporation causes the transient formation of pores, keeping the cells at a lower temperature following the pulse may allow the pores to remain open longer to allow more uptake of the transfectant.

One option is to include pre-chill and post-chill steps by placing the cells/cuvettes on ice or in the refrigerator for a few minutes before and after electroporation.

  • Make sure you wipe off any condensation on the outside of the cuvettes before placing it in the safety stand, safety dome or PEP.
  • The standard electroporation pulse voltage used for cells at room temperature will need to be approximately doubled for electroporation at 4 ͦC to effectively permeate the cell membrane.

Example: If you typically electroporate cells at 250 V at room temperature, then you would want to electroporate the same cells at 500 V at 4 ͦC.

Note: temperature can have big effects on outcome of your experiments. If you are not certain which temperature will work best for your cells it might be a good idea to test a few replicates of different temperature conditions in a single experiment such as room temperature, chilled to 4 ͦC, and chilled on ice side by side with the same electroporation parameters, cells, and transfectant kept constant.

Come back next time when we look at Episode 5 - Considering Transfectant Amount


Click here to visit our Protocol Database, for electroporation protocols searchable by, system, cell/tissue type, application/transfectant, and citation.