Eppendorf Multiporator - Electroporation - Manual (Page 15)

39
4.1.3.2 Testing the tolerance to hypoosmolar conditions
The cells are incubated in hypoosmolar electroporation buffer for 30 minutes at room temperature.
After incubation, the survival rate of the cells is determined by viability staining.
– Trypan blue: Stains dead cells under the microscope.
– Propidium iodide: Stains dead cells under the fluorescence microscope.
If the survival rate of the cells is >90 %, the hypoosmolar buffer can be used in undiluted form for electroporation.
If more than about 10 % of the cells are lysed, the optimal osmolarity of the electroporation buffer must be determined.
In a series of experiments, the cells are incubated for 30 minutes in buffers with a gradually increasing osmolarity.
These buffers are produced by mixing different volumes of the hypoosmolar and the isoosmolar electroporation buffer.
We recommend testing osmolarity according to Table 1.
Table 1
Volumes of Eppendorf Hypoosmolar and Isoosmolar Electroporation Buffers to be used to adjust the desired osmolarity
(final volume: 10 ml).
With the aid of the subsequent viability staining, the optimal osmolarity of the electroporation buffer can be determined.
The lowest osmolarity at which a survival rate of
≥ 90 % is achieved should be used for the following electroporations:
4.1.4 Determining the diameter of the cell
As the size of the cell is a crucial factor for setting the parameters on the Multiporator ® , it should be estimated after
an incubation of 10 to 15 minutes in the electroporation buffer. The most precise measurements can be performed with
electronic instruments, such as the Coulter Counter or Schärfe CASY. As an alternative, the cell diameter can be
estimated under the microscope. This can be performed with the aid of a measuring eyepiece or can be roughly
approximated with the aid of Neubauer's counting chamber or a microgrid.
After the cell diameter has been estimated, the minimum pulse voltage at which the cell membrane can be permeated
can be read from Table 2. The optimal pulse voltage for the electroporation experiment may be 2 to 3 times higher for
suspension cells and 2 to 5 times higher for adherent cells.
Desired osmolarity
Eppendorf
Hypoosmolar Buffer (ml)
Eppendorf
Isoosmolar Buffer (ml)
90 mOsmol/kg 10 0
150 mOsmol/kg 6.8 3.2
200 mOsmol/kg 4.2 5.8
250 mOsmol/kg 1.6 8.4
280 mOsmol/kg 0 10
4 Electroporation protocol
4 Electroporation protocol
Multipor_Appli_E_poration_en.fm Seite 39 Montag, 30. Januar 2006 2:17 14