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Optimizing electroporation parameters

batch to batch. It must be pure enough to ensure that, at 280 mOsmol/kg in bidistilled water, a conductivity of 10 S/cm is not exceeded. The conductivity of individual myo-Inositol batches should be measured before the preparation of the buffer.

Ideally, electroporation should be carried out in hypoosmolar buffer, in which the cell absorbs water shortly before the pulse and then swells up as a result. A number of effects, including a decreased optimal pulse voltage ensure that the plasma membrane can be permeated more easily.

For most cell types, the 2030-minute incubation period in hypoosmolar buffer has no effect on the viability of the cells. However, the incubation in hypoosmolar buffer may induce apoptosis, or even lysis, in very sensitive cells. Therefore, it is strongly recommended to test the tolerance of the cells to hypoosmolar conditions. The easiest way of doing so is by incubating the cells for 30 minutes in hypoosmolar buffer and then performing a viability stain using trypan blue or propidium iodide. If observation under a microscope reveals lysis in more than 10% of the cells, the osmolarity of the buffer must be increased by adding isoosmolar buffer. To determine the optimal osmolarity, it is advisable to incubate the cells in different mixing ratios of hypo- and isoosmolar buffer for 30 minutes prior to the experiment (see Table 2).

This 30-minute period is the maximum incubation time for the cells in the electroporation buffer system. A new viability test followed by observation under a microscope determines the osmolarity that can be tolerated by the cells. The mixing concentrations can then be used for all subsequent experiments with this cell type.


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