Eppendorf Multiporator - Electroporation User Manual

®

Multiporator

Basis-Applikations-Anleitung · Basic Applications Manual

Elektroporation · Electroporation

Inhalt / Table of contents

Basis-Applikations-Anleitung ...............................................................................................................................................3

Basic Applications Manual .................................................................................................................................................27

Nachdruck und Vervielfältigung – auch auszugsweise – nur mit Genehmigung.

No part of this publication may be reproduced without the prior permission of the copyright owner.

Copyright® 2006 by Eppendorf AG, Hamburg

Contents

1 Introduction

1.1 Purpose of the applications guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

2 Principle of the Multiporator®. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

3 Optimizing the parameters

3.1 Optimizing the field strength . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3.2 Length of the field pulse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.3 Number of field pulses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.4. Adjustment of the electroporation buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

3.5 Influence of DNA/RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.6 Influence of the temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.7 Influence of the cell density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

4 Electroporation protocol

4.1 Preparing the cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.1.1 Mycoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.1.2 Cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.1.3 Setting the osmolarity of the electroporation buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.1.3.1 Harvesting adherent cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.1.3.2 Testing the tolerance to hypoosmolar conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.1.4 Determining the diameter of the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

4.1.5 Preparing the DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4.1.6 Selecting the temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40

4.2 Electroporation procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.3 Follow-up treatment of the cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.4 Determination of transfection efficiency in the cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Contents

5 Troubleshooting

6 Ordering information

7Ordering information for North America

8 Appendix

8.1 Guide to determining the minimum voltage to be set on the Multiporator

8.2 Volumes of hypoosmolar and isoosmolar electroporation buffer for setting required osmolarity (10 ml) . . . . 49

8.3 Composition of electroporation buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

8.4 Protocol for the electroporation of eukaryotic cells, based on Jurkat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47

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. . . . . . . . . . . . . . . . . . . . . . . . . . 48

27

1 Introduction

The phenomenon whereby a short, intensive current surge (pulse) is used to generate reversible openings (pores) in a
membrane was first used in the 1970s to introduce foreign molecules into cells. These membrane pores allow low­molecular substances (such as dyes or peptides) and high-molecular substances (such as proteins, DNA and RNA) to be
introduced into cells. This procedure, which is known as electroporation, electroinjection or electropermeabilization, has
developed into a standard method in many laboratories, and is used primarily for the transfection of eukaryotic cells and
bacteria.

1 Introduction

The Eppendorf Multiporator
place efficiently and gently under hypoosmolar conditions. The hypoosmolar buffer causes the cells to swell up, which
expands the membrane and loosens up the cytoskeleton. This in turn leads to a reduction in voltage required for the
formation of membrane pores. Electroporation can thus be performed in a more "cell-friendly" manner without any
adverse effect on transfection efficiency.

1.1 Purpose of the applications guide

This guide is valid for the eukaryotic module of the Multiporator, by which eukaryotic cells (except yeast and some
microorganisms) can be electroporated.

It contains concise descriptions of the experimental conditions that form the basis of the innovative electroporation
process carried out using the Multiporator's Soft Pulse technology. Time should be taken to familiarize oneself with the
effects that important parameters of the Soft Pulse technology, such as pulse voltage, time, temperature and the
composition of the medium, have on the transfection yield. This will enable the best possible transfection results to be
achieved with a specific cell line.

®

and the special electroporation buffers form a system which allows electroporation to take

Any experiences with commonly used electroporation techniques in the past utilizing millisecond pulse times cannot be
compared directly to the technology using the Multiporator

Application protocols for many different cell lines can be found on the Eppendorf homepage at www.eppendorf.com.
If cells are used for which no application protocol is available, Section 3, "Optimizing the parameters", and the general
electroporation protocol in Section 4 should be consulted. Section 5, "Troubleshooting", contains assistance for those
occasions when results have not turned out as expected.

Bacteria, yeast as well as other microorganisms can be transformed with the optional bacteria module. Application
protocols are available at www.eppendorf.com too.

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with microsecond pulse times.

28

2 Principle of the Multiporator®

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The electroporation functions of the Multiporator
devices. The main differences are as follows:

– Application of extremely short pulses, in the range of 15 to 100 microseconds, versus pulses in the millisecond range.

– Electronic pulse regulation, which allows uniform, reproducible pulses regardless of the resistance properties of the

media used.

– Electroporation in a hypoosmolar buffer which is non-toxic and which is adapted to the cytosolic ion composition of

the cells.

The combination of these features guarantees high transfection yields without severe damage to the cells. This contrasts
sharply with observations frequently made during electroporation in the millisecond range.

Soft Pulses

During electroporation, the membrane of a cell is charged up to a voltage at which the cell membrane is (reversibly)
permeated. The pulse lengths (i.e. the time constant τ) used with the Multiporator
between 15 µs and 100 µs. In this period, the membrane is permeated when the permeation voltage is exceeded. This
leads to a drastic increase in the permeability of the membrane, which can be considered as pore-like openings in the
membrane.

If the external voltage applied is up to one thousand times longer (as is the case with milliseconds), high electrical
currents flowing through the inside of the cells inflict severe damage upon the cells themselves. Irreversible damage can
be caused to the membrane functions and the genome, as well as irreversible changes in the ion composition inside of
the pulsed cells. This is the situation with many conventional electroporation devices.

When the Soft Pulse technology of the Multiporator
a breakthrough of the membrane occurs. The exponentially decaying pulse prevents significant amounts of current from
flowing through the cells after the pores have formed.

In addition, the Soft Pulse is measured continuously by the Multiporator
electronic regulation enables extremely high reproducibility.

are fundamentally different to those of other commercially available

®

for plant and animal cells are usually

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is applied, however, the cell is charged up only to the point at which

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and re-regulated every 5 µs. This unique

2 Principle of the Multiporator

®

As a result, the Multiporator® ensures extremely high transfection rates.

Multiporator® buffer system

Pulse media with low electrical conductivity ensure that the current flow is markedly reduced during electroporation, thus
preventing any significant damage to cells. In addition, such media guarantee that the electrically induced "pores" are
much larger than those obtained from pulses in conductive solutions, such as phosphate-buffered saline solutions (1).

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The Multiporator

Shifts in the pH value

If the current flow lasts a long time, electrolysis of water takes place on the electrodes of the cuvette. When millisecond
pulses are used, the pH value directly at the electrodes changes drastically. Shifts of the pH value in the alkaline and in
the acidic range are non-physiological and damage the cells. In contrast, no significant changes in the pH values in and
around the electrodes are noted when the Multiporator

is specially designed for the use of such low-conductivity pulse media.

®

is used (2).

29

2 Principle of the Multiporator®

Release of aluminum during electroporation

Commercially available disposable cuvettes usually contain aluminum electrodes. Aluminum is released from the
electrodes under both acidic and alkaline conditions. The shift in the pH value inside the cuvette, which can be observed
after millisecond pulses, leads to a release of large quantities of cytotoxic aluminum ions into the pulse medium. The
Multiporator's Soft Pulses prevent any cell-damaging increase in the aluminum concentration in the cuvette (2).

Hypoosmolar conditions

The hypoosmolar pulse medium contains far fewer osmotically active substances than culture media or physiological
buffer solutions, such as PBS. In a hypoosmolar medium, the cell absorbs water and swells up. Both the cell and its
nucleus assume a rounded form and the cell membrane becomes detached from the cytoskeleton, thereby greatly
facilitating electroporation of the cell.

+

/ K+ gradient of the cell

Na

2 Principle of the Multiporator®

Eukaryotic cells build up a gradient in the concentration of sodium and potassium ions across the cell membrane. When
electroporation has caused the membrane to become highly permeable, (particularly for small ions), the Na+/K+ gradient
breaks down locally. The presence of sodium ions in the electroporation buffer (in PBS, for example) makes the situation
even worse, since Na
prevent sodium from entering the cell and, moreover, stops the K+ gradient from collapsing completely.

The synergy of the Multiporator

The quality of the Multiporator's performance is derived from the combination of device and pulse medium. The
Multiporator® applies Soft Pulses, which lead to a gentle permeation of the cell membrane. The electronically regulated
voltage curve guarantees high reproducibility. In combination with the Multiporator
fulfills several different functions.

+

ions enter the cell. With K+ as its sole cation, the special Multiporator® electroporation buffers

®

and the buffer system

®

, the special electroporation buffer

1. Low electrical conductivity prevents high current flow and the resulting changes in pH values and therefore prevents
an increase in the release of cytotoxic aluminum.

2. The low osmolarity of the pulse medium enables the cell to swell up and round off, thus enabling an easier and more
controlled electroporation.

+/K+

3. The ion composition of the buffer maintains the Na

The synergy of the device and the pulse medium is a result of these fundamental electroporation factors being taken into
consideration.

gradient of the cells.

30

2 Principle of the Multiporator®

Biophysical basics of the Multiporator's technique

Crucial parameters for successful electroporation are the voltage and the length of the pulse (time constant τ) used.
Both factors can be set directly on the Multiporator®. The parameters for major applications can be found in numerous
existing application protocols, available on the Eppendorf homepage at www.eppendorf.com. For a new application,
reference values for the optimal pulse voltage can be calculated or can be taken from the corresponding tables
(see Sections 3.1 and 4.1.4).

Voltage and pulse length

[V]

1000

800

5 µs

5 µs

600

400

200

37%

0

04080120 160

Fig. 1: Controlled exponential decay of the voltage of the microsecond pulse of the Multiporator

()τ

200

[µs]

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2 Principle of the Multiporator®

The pulse voltage is readjusted every 5 µs. After the time constant τ (tau), the pulse voltage has dropped down to 37 %
of its initial value. The initial pulse voltage and the time constant τ are the only parameters which define the microsecond
pulse of the Multiporator®.

®

The voltage set on the Multiporator
time constant (τ) shown is the time required for the voltage to decrease to the value V0/e (= approximately 37 % of the
initial voltage). For example, if a voltage of 1,000 V and a time constant of 40 µs have been set on the Multiporator®, the
initial voltage of the Soft Pulses is 1,000 V. After 40 µs, this voltage has decreased to approximately 370 V. After these
40 µs, the electronic control of the Multiporator

Gap width and field strength

Since the strength of an electrical field depends on the distance of the electrodes, the usage of electroporation cuvettes
with a gap width of 1 mm, 2 mm, or 4 mm results in a different field strength (= pulse voltage [V]/gap width [cm]) in the
cuvette. As the 100 µl volume of the 1-mm cuvette is extremely small, electroporation of eukaryotic cells is normally
carried out using cuvettes with a gap width of 2 mm (400 µl) or 4 mm (800 µl). If a voltage of 800 V is set on the
Multiporator®, a field strength of 2,000 V/cm is produced when a 4-mm cuvette is used. However, if a cuvette with a gap
width of only 1 mm is used at the same setting, the field strength is 8,000 V/cm! For this reason, particular attention must
be paid to the type of cuvette used for the experiments.

corresponds to the initial voltage (V0) of the discharge curve shown in Fig. 1. The

®

will have regulated the voltage eight times (i.e. at intervals of 5 µs).

31

2 Principle of the Multiporator®

Calculating the field strength

The critical field strength which is necessary for electropermeation of the membrane can be calculated approximately. To
do so, a rough determination of the diameter (d) of the cell must be made. Based on the determined diameter, the critical
field strength can be calculated using the following formula:

Ec = Vc / (0.75 x d)

Ec Critical field strength [V / cm]
V

Permeation voltage of the membrane [1 V at 20 °C/ 2 V at 4 °C]

c

d

Cell diameter [cm]

The following is an example for a cell with a diameter of 20 µm (2 x 10-3 cm) at room temperature:

Ec = 1 V / (0.75 x 2 x10

Ec = 667 V/cm

2 Principle of the Multiporator®

To calculate the voltage which has to be set on the Multiporator®, it is necessary to multiply the field strength EC by the
gap width of the cuvette. In our example, a minimum voltage of 667 V/cm x 0.2 cm = 133 V must be set for a 2-mm
cuvette. For a 4-mm cuvette, twice this value (667 V/cm x 0.4 cm = 267 V) is required.

-3

cm)

When electroporation is carried out at 4 °C, the E
VC= 2 V at 4 °C).

At the critical field strength EC, pores form on the poles of the cells oriented in the field direction which small molecules
or ions can pass through. It is possible to test "pore formation" immediately using propidium iodide. The red
fluorescence of this dye can be detected when it has been incorporated into the cell and has bound to nucleic acids.
However, for large molecules, such as nucleic acids, the values used must be higher than the critical field strength E
In the case of suspension cells, the ideal value for introducing plasmid DNA into the cell is normally 1 to 3 times that of
EC. For adherent cells, a value 1 to 5 times that of EC is necessary to introduce DNA into the cell.

! Eppendorf has step-by-step application protocols for the Multiporator® for many frequently used cell lines,
which can be found on the Eppendorf homepage at www.eppendorf.com !

Duration of the Soft Pulse

As aforementioned, microsecond pulses are ideal for highly efficient, gentle electroporation. A general rule is that large
cells require a longer time for reversible membrane permeation. With the Multiporator
between 5 µs and a maximum of 100 µs are normally used for electroporation. These times are tailored to suit the
Multiporator® buffer system.

An optimization strategy for new applications with regard to all relevant parameters (e.g. field strengths, pulse lengths)
is described in detail in the following section.

value is twice as high as the value at room temperature (because

C

®

, pulses with a time constant

.

C

Bibliography

1) Sukhorukov, V.L., Mussauer, H. and Zimmermann, U. (1998) The effect of electrical deformation forces on the
electropermeabilisation of erythrocyte membranes in low- and high conductivity media. J.Membr. Biol. 163, 235-245.

2) Friedrich, U., Stachowicz, N., Simm, A., Fuhr, G., Lucas, K. and Zimmermann, U. (1998) High efficiency electrotrans­fection with aluminium electrodes using microsecond controlled pulses.
Bioelectrochemistry and Bioenergetics 47, 103-111.

32

3 Optimizing the parameters

To ensure that maximum transfection rates are achieved, the electroporation parameters should be optimized for each
new cell line. This section contains guidelines for determining the ideal parameters as simply and as quickly as possible.

3.1 Optimizing the field strength

The field strength (V/cm) of the electrical pulse used is an essential factor in determining the survival rate as well as the
transfection rate of the cells used.

If the field strength of the pulse exceeds a characteristic value (= critical external field strength), reversible permeation
occurs in the cell membrane. This so-called permeation voltage is heavily dependent on the cell diameter and the
temperature at which electroporation takes place. The diagrams in Fig. 2 show the permeation voltage that has to be set
in relation to the cell diameter and the temperature at which the electroporation is performed. The diameter of the cell is
determined after the cells have been incubated in electroporation buffer for 10 – 15 minutes (see Sec. 3.4 + 7.1).

In addition, the gap width of the cuvettes must be taken into account when the minimum pulse voltage is determined.
If the gap width is doubled, the pulse voltage must also be doubled in order to obtain the same field strength. A general
rule when determining the ideal field strength is that small cells require a higher field strength in order to achieve
membrane permeation. The pulse voltages in Fig. 2 and Table 2 (page 16) are the minimum values at which the
membrane can be permeated. However, depending on the cell type used, optimal transfection efficiency is often only
achieved at significantly higher voltages. To determine the optimal pulse voltage, it is advisable to carry out a series of
experiments in which the minimum value, twice the value and then three times the value shown in Table 2 (page 16) are
used for suspension cells, and up to five times the value for adherent cells. Cells which do not assume a rounded form in
the electroporation buffer often require even higher pulse voltages before optimal transfection can occur.

Please note that increasing the pulse voltage can increase the transfection rate but, at the same time, can also increase
the cell mortality rate.

3 Optimizing the parameters

Cuvette: 2 mm gap width

1400

400 µl volume

4°C

1200

1000

800

600

Voltage [V]

400

200

0

RT

Voltage [V]

020406080100

Cell diameter [µm]

Fig. 2: Minimum pulse voltage at which the cell membrane is permeated

Cuvette: 4 mm gap width

2800

2400

2000

1600

1200

800

400

0

800 µl volume

4°C

RT

020406080100

Cell diameter [µm]

33

3 Optimizing the parameters

The minimum pulse voltage is dependent on the cell diameter following incubation in electroporation buffer as well as on
the temperature and on the gap width of the cuvettes. The values shown can be used to determine the ideal pulse
voltage to be set on the device, as described in Sec. 3.1.

Example

Cell type: Suspension cells
Cell diameter in electroporation medium: ~ 20 µm
Gap width of cuvette: 2 mm
Temperature in the cuvette: Room temperature
Minimum pulse voltage according to diagrams: ~ 130 V
Series of experiments for optimizing pulse voltage: 130 V / 260 V / 390 V

3 Optimizing the parameters

3.2 Length of the field pulse

In addition to the field strength, a crucial factor for successful transfection is the pulse length.

The length of the pulse is primarily dependent on the diameter of the cell: the larger the cell, the longer the pulse
necessary for permeation of the membrane.

Empirically, the ideal pulse lengths for electroporation have proved to be 40 to 100 µs at room temperature and
15 to 40 µs at 4 °C. To optimize the duration of the pulse, three different pulse lengths should be selected within the
above-mentioned ranges.

3.3 Number of field pulses

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 to achieve the desired result, with a 60-second
interval between pulses to allow the cell membrane to regenerate. During multiple pulsing, the Multiporator
maintains this interval between each pulse. An intact and resealed cell membrane is a prerequisite for the build-up of the
membrane potential, which is essential for electroporation. In addition, the cells rotate during this regeneration phase
(Brownian movement), which virtually rules out the danger of further "injury" being caused to the same membrane area by
a second pulse.

3.4. Adjustment of the electroporation buffer

®

A precondition for successful electroporation with the Multiporator
conductivity.

The best possible transfection results are obtained by using the original buffers from Eppendorf, which have been tested
for sterility as well as for the absence of mycoplasma, endotoxins and pyrogen and which have also undergone a
thorough cytotoxicity test.

However, users may also make up this buffer themselves (see Sec. 7.3).

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 permeation voltage, ensure that
the plasma membrane can be permeated more easily.

is the special buffer system with low electrical

®

automatically

34

3 Optimizing the parameters

For most cell types, the 20- to 30-minute incubation period in hypoosmolar buffer, which is unavoidable due to the
conditions of the experiment, 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 1, page 13). 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.

Irrespective of the buffer system selected, it is essential to ensure that the cells do not remain in the electroporation
buffer for longer than 30 minutes.

3.5 Influence of DNA/RNA

The transfection efficiency of electroporation can be affected by the concentration, the purity and the size of the
molecules used.

a) Influence of nucleic acid concentration

With the optimal electroporation parameters (osmolarity, voltage, pulse length), the quality of the results obtained at
plasmid concentrations between 5 µg/ml and 20 µg/ml is usually satisfactory.
The efficiency of the transfection may be raised by increasing the nucleic acid concentration, but only within a limited
concentration range. Tests with various different cell lines have shown that only in very few cases (e.g. when large
plasmids were used) plasmid concentrations in excess of 20 µg/ml lead to an increase in the transfection rate
(see Fig. 3).

3 Optimizing the parameters

100

50

0

ransient EGFP-N1 expression [%]

T

Fig. 3: Transient transfection efficiency in NIH-3T3 cells in relation to the DNA concentration (µg/ml).

The cells were electroporated with different concentrations of the pEGFP-N1 plasmid.
The transfection rate (max. = 100 %) was determined by FACS analysis.

010203040

Plasmid DNA [µg/ml]

35

3 Optimizing the parameters

b) Influence of nucleic acid purity

Empirical studies have shown that EDTA and buffer salts such as HEPES or TRIS can drastically reduce transfection
efficiency. We therefore recommend dissolving the nucleic acid in distilled water before transfection. Any losses
resulting from DNA buffer exchange are usually more than compensated for by the increased transfection efficiency.
Irrespective of the preparation method used, the DNA/RNA should be ultra-pure (A
It is also important to use endotoxin-free DNA. Otherwise an increase in the DNA concentration will lead to an
increase in the endotoxin content in the cell suspension.

c) Influence of the size of the plasmid

Transfection efficiency is also affected by the size of the individual molecule that is introduced into the cell. This
means that the optimal electroporation parameters that were determined for a certain plasmid, for example, have to
be changed when a larger or smaller plasmid is used.

260

nm/A

3 Optimizing the parameters

3.6 Influence of the temperature

The temperature has a direct effect on the permeation voltage of the cell membrane as well as on the regeneration of the
membrane following electroporation.

a) Influence of the temperature on the permeation voltage of the cell membrane

Since the permeation voltage at 4 °C is twice that at room temperature, it is essential to take the temperature into
account when determining the optimal field strength of the pulse. Therefore, during electroporation at 4 °C, the
necessary field strength of the pulse is also nearly twice as high as those values for room temperature. However,
mammalian cells are usually electroporated at room temperature.

nm ≥ 1.8 ).

280

b) Influence of the temperature on the regeneration of the cell membrane

Incubating cells following electroporation at low temperatures (e.g. 4 °C) slows down the healing process of the cell
membrane. In the case of eukaryotic cells, the resealing of the membrane pores can take half an hour and longer
under these conditions. With certain cell types, this can lead to an increase in the amount of transfection material
absorbed. However, some cells are extremely sensitive to low temperatures, particularly when permeated, and can
suffer from irreversible damage after short incubation times in a cold environment.
In those cases where electroporation at 4 °C leads to higher transfection rates, the cells' chances of survival can be
boosted if they are resuspended in electroporation buffer at 37 °C or room temperature, cooled down to 4 °C and
then transferred into precooled cuvettes. Following electroporation, the cells are incubated at 4 °C for a maximum of
two minutes and then heated to 37 °C.
Electroporation at higher temperatures (e.g. >25 °C) causes the permeated membrane areas to seal up more rapidly,
which accelerates membrane regeneration and thus increases the cell survival rate. However, the transfection rate
may be lower than that obtained when electroporation is carried out at low temperatures.

c) Influence of the temperature on the conductivity of the medium

The temperature has a profound effect on the conductivity of the electroporation buffer. Increasing the temperature
causes the conductivity of a solution to increase as well, which may lead to lower transfection rates. For this reason,
it is advisable not to work at temperatures in excess of 33 °C.

36

3 Optimizing the parameters

3.7 Influence of the cell density

6

A cell density of 1 x 10
in this range. When high cell densities (>3 x 10
i.e. the cells are no longer evenly exposed to the electrical field. This may lead to cell fusion and cause the transfection
rate to decrease.

However, using a cell density of <1 x 10

cells/ml is recommended for electroporation since the electrical field is still effective on the cells

6

) are used, homogeneous field conditions can no longer be guaranteed,

6

cells/ml should have no negative effect on the transfection rate.

3 Optimizing the parameters

Bibliography

3) Zimmermann, U., Effect of high intensity electric field pulses on eucaryotic cell membranes.
In: Effect of high intensity electric field pulses on eucaryotic cell membranes.
U. Zimmermann and G. Neill, editors. pp. 1-106. CRC Press, Boca Raton, 1996.

37

4 Electroporation protocol

Cell-specific application protocols are available on the Eppendorf homepage at www.eppendorf.com. The list of
applications is updated on a regular basis. (A protocol for Jurkat is included in Appendix 8.4).

If no application protocol is available for the examined cell type, the following general guidelines for the electroporation
of eukaryotic cells can be used.

To obtain the best possible transfection results, we recommend determining the optimal electroporation parameters in an
experiment. Information on how to optimize the parameters can be found in Sec. 3, "Optimizing electroporation
parameters".

4.1 Preparing the cells

4 Electroporation protocoll

4.1.1 Mycoplasma

Mycoplasma prevent efficient and reproducible electroporation of cells. Therefore it is essential to test the cells for the
presence of mycoplasma. There are several tests available.

One common method is the DNA fluorochrome staining. This test is based on DNA staining using Hoechst dye 33258 or
DAPI, which makes mycoplasma-specific DNA visible under a fluorescence microscope.

The most sensitive current method for detecting mycoplasma is by PCR. PCR detection kits are commercially available
but are more time-consuming and expensive than the aforementioned methods.

4.1.2 Cell culture

When electroporation occurs, the cells should already have passed several growth cycles. No freshly thawed or recently
transported cells should be used since this additional stress would have a negative effect on the transfection rate.

The cells should be in the exponential growth phase when transfection takes place.

4.1.3 Setting the osmolarity of the electroporation buffer

®

Electroporation with the Multiporator
of the cells to hypoosmolar conditions has to be tested in a preliminary experiment (see Sec. 3.4).

4.1.3.1 Harvesting adherent cells

Cells should be harvested as gently as possible. According to the cell type, the following methods can be used:

Dispase (concentration: 0.01 to 0.1 % w/v). This has proved to be the most gentle method for harvesting.

•

Trypsin (HPLC-grade, without EDTA, 0.1 to 0.25 % w/v). Prior to the addition of trypsin, the cells must be washed at

•

least twice with PBS, without Ca2+ and Mg2+.

•

Scrape the cells carefully from the bottom of the culture dish.

should ideally be carried out in hypoosmolar electroporation buffer. The tolerance

38

4 Electroporation protocol

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).

Eppendorf

Desired osmolarity

90 mOsmol/kg

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

Hypoosmolar Buffer (ml)

10 0

Eppendorf

Isoosmolar Buffer (ml)

4 Electroporation protocol

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
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.

, it should be estimated after

39

4 Electroporation protocol

Table 2

Minimum pulse voltages at which the cell membranes may be permeated, in relation to the diameter of the cell after
incubation for 10 to15 minutes in the electroporation buffer, the electroporation temperature and the gap width of the
cuvette. Depending on the cell line, the optimal pulse voltage for the electroporation experiment may be between two
and five times higher.

Voltage

Diameter of cell

[µm]

5 530 1100 1100*

4 Electroporation protocol

* The maximum voltage which can be applied with the eukaryotic module of the Multiporator® is 1,200 V.

4.1.5 Preparing the DNA

The DNA should be ultra-pure (A260/A280 ≥ 1.8 ) and endotoxin-free. Following the last purification step, it should be
resuspended directly in bidistilled water and not in TE buffer.

10 270 540 540 1100

15 180 360 360 710

20 130 260 260 530

25 110 220 220 430

30 90 180 180 360

35 80 160 160 310

40 70 140 140 270

45 60 120 120 240

50 50 100 100 210

60 40 80 80 160

80 30 60 60 120

2-mm cuvette

room temp.

Voltage

4-mm cuvette

room temp.

Voltage

2-mm cuvette

4 °C

Voltage

4-mm cuvette

4 °C

*

4.1.6 Selecting the temperature

Electroporation may be carried out at room temperature or at 4 °C (see Sec. 3.6). Experiments to establish new protocols
normally take place at room temperature.

However, if electroporation takes place at 4 °C in order to achieve higher transfection rates, the cells should not remain
on ice for more than two minutes before being incubated at 37 °C.

40

4 Electroporation protocol

4.2 Electroporation procedure

Electroporation conditions must be optimized for every cell line for which no specific application protocol is available.
The following protocol is a general guideline for the electroporation of eukaryotic cells. To determine the optimal
electroporation conditions for highest transfection efficiency, please refer to Section 3.

1. Ensure that cells are harvested in the exponential growth phase.

2. Dilute the cells in culture medium with 0.5 to 1 % FCS and determine the number of cells and spin the cells down.

3. Resuspend the cells in Eppendorf Electroporation Buffer (at RT or 4 °C) with the determined osmolarity and set a cell
concentration of between 1 x 10

Caution: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to

guarantee successful electroporation!

4. Aliquot the cell suspension (400 µl for a cuvette with 2-mm gap width and 800 µl for a cuvette with 4-mm gap width)
in Eppendorf tubes. Add plasmid DNA (final concentration 5 to 20 µg/ml) or proteins (final concentration
10 to 100 µg/ml) and mix.
When performing electroporation at 4 °C, precool the cuvettes on ice.

5. Transfer the cell suspension to electroporation cuvettes. Take care that no air bubbles are formed.

6. Electroporation: (settings on the Multiporator

Mode

: Eukaryotic cells

oltage (U): To enable the optimal voltage to be set on the Multiporator®, it is advisable to perform a series

V

of experiments with several pulse voltages.
For adherent cells: between 1 to 5 times the minimum pulse voltage stated in Table 2.
For suspension cells: between 1 to 3 times the minimum pulse voltage stated in Table 2.

Time constant (

Number of pulses

τ): At RT 40 to 100 µs

At 4 °C 15 to 40 µs

(n): 1

6

and 3 x 106 cells/ml, or slightly lower.

®

)

4 Electroporation protocol

7. After pulsing, allow the cell suspension to remain in the cuvette for 5 to10 minutes.
If electroporation was carried out at 4 °C, the cuvettes should be placed on ice for a maximum of 2 minutes after
pulsing and should then be incubated in a water bath for 8 minutes at 37 °C.

8. Carefully remove the cell suspension from the cuvette using a Pasteur pipette and cultivate it in 3 to 5 ml culture
medium in a 60-mm culture dish.
When removing the cell suspension, ensure that the aluminum electrodes are not damaged so that contamination by
cytotoxic aluminum ions is prevented.
Note: After pulsing, the cells should be incubated for 2 to 3 hours at 37 °C before any centrifugation is performed, to
ensure resealing of the membrane.

4.3 Follow-up treatment of the cells

After the cells have been transferred to the culture medium, they should not be subjected to stress, such as can be
caused by shaking or long periods of transport.

4.4 Determination of transfection efficiency in the cells

Depending on the cell type and on the plasmid used, transient expression may be detected roughly 24 to 48 hours after
transfection has taken place. In some cases (e.g. primary cells), this may require considerably longer.

41

5 Troubleshooting

If the transfection experiments do not turn out quite as expected, helpful information may be found in the following
troubleshooting guide.

The specific problems listed are caused by a variety of factors which the researcher can easily narrow down by
optimizing specific protocol areas, as described briefly below and outlined in full in Sec. 3 and 4.

Problem Possible cause Solution / comments

5 Troubleshooting

Low survival rate Pulse is too strong. Check determination of the cell size (Sec. 4.1.4)

and minimum field strength based on this size
(Table 2). Note that gap width of the cuvette and
temperature during electroporation must also
be taken into consideration.

Pulse is too long. Shorten the pulse length to decrease

permeation of the cell membrane. This can
increase the survival rate of the cells. Note that
the optimal pulse length is affected by the
temperature at which electroporation is carried
out (Sec. 3.6).

Too many pulses are applied. Reduce the number of pulses. Multiple

permeation of the same membrane areas can
lead to irreversible damage to the plasma
membrane.

Conductivity of the electro­poration buffer is too high.

Cells remained too long in
the electroporation buffer.

Check the conductivity of your electroporation
buffer using a suitable measuring device.
Buffers with a conductivity of >4mS/cm can
lead to a reduced incorporation rate. Low­conductivity Eppendorf electroporation buffers
are recommended. The addition of plasmid
DNA dissolved in a buffer solution instead of
distilled water can also increase conductivity.

If overall incubation of the cells in the
electroporation buffer exceeds 30 minutes,
apoptosis maybe inducted in certain cell types.
Shorten the duration of the experiment by
carrying out individual steps more quickly,
by shortening the washing procedure prior to
pulsing, or by cutting the incubation time of the
cells after the pulse. (Attention: An incubation
time of 5 to 10 minutes at room temperature
should be maintained. Then transfer the cells
into culture medium and cultivate at 37 °C).
Before any centrifugation is performed after
pulsing, the cells should be incubated for 2 to
3 hours at 37 °C to ensure resealing of the cell­membrane.

42

5 Troubleshooting

Problem Possible cause Solution / comments

5 Troubleshooting

Gene product has
a toxic effect on the cell.

After pulsing, cells are incubated
too long at a low temperature
(4 °C).

The cells are damaged during
the harvesting procedure.

Osmolarity of the electro­poration buffer is too low.

Cells are stressed. At the time of electroporation, the cells should

The gene product itself or a high expression rate
of the gene product may have a toxic effect on
the cells. The optimal plasmid concentration
should be tested individually for each cell type
and each plasmid.

An excessive incubation period on ice can lead
to cell death. The incubation period on ice
should not exceed two minutes.

Using trypsin with an excessively high concen­tration or with an insufficient purity level during
the harvesting of the cells may increase the
mortality rate. Dispase or trypsin HPLC-grade
(Sec. 4.1.3.1) is recommended.

Low osmolarity of the electroporation buffer
may cause sensitive cells to swell to such an
extent that they burst during electroporation.
This effect can be tested by a viability staining
of the cells 2–3 hours after electroporation
(Sec. 4.1.3.2). Increase the amount of
isoosmolar electroporation buffer to raise the
osmolarity of the buffer and repeat the viability
test.

have been in culture for several cycles. Freshly
thawed or recently transported cells are still in a
condition of stress and should not be used
immediately for electroporation.

Low transfection rates Cells are contaminated

with mycoplasma.

Pulse is too weak. A pulse with a low field strength may be too

Mycoplasma prevent successful electroporation
of cells with the Multiporator
must be checked for mycoplasma at regular
intervals (Sec. 4.1.1).

weak to permeate the cell membrane. Check
determination of the cell size (Sec. 4.1.4) and
the minimum field strength based on this size
(Table 2). The gap width of the cuvette and the
electroporation temperature must also be
considered.

®

. Cell cultures

43

5 Troubleshooting

Problem Possible cause Solution / comments

Pulse is too short. Extend the pulse length to increase permeability

of the cell membrane, which can lead to a
higher transfection rate. Note that the optimal
pulse length is affected by the temperature at
which electroporation is carried out.

5 Troubleshooting

DNA concentration
is too low / too high.

Large plasmids are used. The size of the plasmid can greatly affect the

Cell density is too high. If cell densities are too high during

Electroporation preparation
contains EDTA or endotoxins.

If the transfection rate is too low and the viability
of the cells is high, the plasmid concentration
may be increased.
Note, however, that an increased plasmid
concentration may lead to a higher transfection
rate only within a limited concentration range of
the plasmid.

transfection rate. When large plasmids are
used, it may be necessary to increase the
permeation of the membrane by applying
pulses with a higher field strength. Be careful as
this may also lead to a higher cell mortality rate.

electroporation, the homogeneity of the
electrical field can no longer be guaranteed.
Reduce the cell concentration in the cuvette to
1 – 3 x 10

EDTA and endotoxins have a cytotoxic effect.
They are often introduced into the electropora­tion preparation when DNA is added (e.g. in
TE buffer). Remove EDTA by carrying out a
buffer exchange on bidistilled H
can be removed using "endotoxin-free" plasmid
preparation kits.

6

cells/ml or lower.

0. Endotoxins

2

Incubation period for gene
expression is too short.

Problems with
the reporter assay.

Non-reproducible results Washing procedure prior

to electroporation was
not thorough enough.

44

Following transfection, different cell types
require different incubation periods in order to
reach their maximum expression rate.
Expression should be checked again at a later
point in time.

Include positive controls which indicate that the
reporter system is working properly.

Traces of medium can have a severe effect on
the conductivity of the electroporation buffer,
and thus on the transfection result. Therefore,
the medium in the supernatant must be
thoroughly removed during the washing
procedure.

5 Troubleshooting

Problem Possible cause Solution / comments

5 Troubleshooting

Cuvette has been used
several times.

Cells were harvested
at different confluencies.

Conductivity of the electropora­tion buffers varies (such as when
self-prepared buffers are used).

No transfection Pulse was not injected into

the cell suspension due to poor
contact between cuvette and
Multiporator

®

.

Using an electroporation cuvette several times
may result in a non-homogeneous electrical
field during electroporation. Only new cuvettes
are recommended for important experiments.

Harvesting cells at different confluencies can
lead to non-reproducible transfection results.
Cells should always be harvested in the
exponential growth phase.

Check the conductivity of self-prepared buffers
on a regular basis using a suitable measuring
device. A defined conductivity is always
guaranteed with Eppendorf electroporation
buffers.

Check that cuvette and the cuvette insert are
correctly inserted in the Multiporator

®

.

45

6 Ordering information

Order no:

4308 000.015 Multiporator

for eukaryotics

4308 000.023 Multiporator

for eukaryotics, bacteria and yeasts

4308 000.031 Multiporator

for eukaryotics, cell fusion, with 1 Helix fusion chamber and 1 Micro fusion chamber

4308 000.040 Multiporator

for eukaryotics, bacteria, yeast and cell fusion, with 1 Helix fusion chamber and 1

6 Ordering information

4308 070.501 Hypoosmolar buffer (PH), sterile, 100 ml

4308 070.510 Isoosmolar buffer (PI), sterile,100 ml

4308 070.528 Hypoosmolar buffer (FH), sterile,100 ml

4308 070.536 Isoosmolar buffer (FI), sterile,100 ml

Micro fusion chamber

Electroporation buffer

Electrofusion buffer

®

®

®

®

Electroporation cuvettes

4307 000.569 1 mm gap width, aluminum, sterile, 50 pcs.

4307 000.593 2 mm gap width, aluminum, sterile, 50 pcs.

4307 000.623 4 mm gap width, aluminum, sterile, 50 pcs.

4308 078.006 Cuvette stand for 16 electroporation cuvettes

4308 021.004 Insert (electroporation / electrofusion)

for connecting to external electrodes

4308 014.008 Helix fusion chamber for cell fusion

(gap between electrodes: 0.2 mm)

4308 030.003 Micro fusion chamber

(gap between electrodes: 0.2 mm)

4308 031.000 Micro fusion chamber

(gap between electrodes: 0.5 mm)

4308 017.007 Stand for 10 helix fusion chambers

4308 010.002 Conversion kit for mode for bacteria

(to be installed by SERVICE)

4308 011.009 Conversion kit for mode for cell fusion

(to be installed by SERVICE)

46

7 Ordering information for North America

Order no:

940000505 Multiporator

for eukaryotics

940000602 Multiporator

for eukaryotics, bacteria and yeasts

940000700 Multiporator

for eukaryotics, cell fusion, with 1 Helix fusion chamber and 1 Micro fusion chamber

940000807 Multiporator

for eukaryotics, bacteria, yeast and cell fusion, with 1 Helix fusion chamber and
1 Micro fusion chamber

Electroporation buffer

940002001 Hypoosmolar buffer (PH), sterile, 100 ml

940002109 Isoosmolar buffer (PI), sterile,100 ml

Electrofusion buffer

940002150 Hypoosmolar buffer (FH), sterile,100 ml

940001021 Isoosmolar buffer (FI), sterile,100 ml

Electroporation cuvettes

940001005 1 mm gap width, aluminum, sterile, 50 pcs.

940001013 2 mm gap width, aluminum, sterile, 50 pcs.

940001021 4 mm gap width, aluminum, sterile, 50 pcs.

®

®

®

®

7 Ordering information for North America

940001102 Cuvette stand for 16 electroporation cuvettes

940004209 Insert (electroporation / electrofusion)

for connecting to external electrodes

940001200 Helix fusion chamber for cell fusion

(gap between electrodes: 0.2 mm)

940001251 Micro fusion chamber

(gap between electrodes: 0.2 mm)

940001234 Micro fusion chamber

(gap between electrodes: 0.5 mm)

940001218 Stand for 10 helix fusion chambers

940004101 Conversion kit for mode for bacteria

(to be installed by SERVICE)

940004128 Conversion kit for mode for cell fusion

(to be installed by SERVICE)

47

8 Appendix

8.1 Guide to determining the minimum voltage to be set on the Multiporator

8 Appendix

1400

Cuvette: 2 mm gap width

400 µl volume

4°C

1200

1000

800

600

Voltage [V]

400

200

0

RT

020406080100

Cell diameter [µm]

Fig. 4a:

®

2800

2400

2000

1600

1200

Voltage [V]

800

400

0

Cuvette: 4 mm gap width

800 µl volume

4°C

RT

020406080100

Cell diameter [µm]

Minimum pulse voltages at which the cell membrane is permeated
The minimum pulse voltage is dependent on the cell diameter following 10- to 15-minute incubation in electroporation
buffer as well as on the temperature and the gap width of the cuvettes. These values, shown as a graph (4a) and as a
table (4b), can be used to determine the optimal pulse voltage to be set on the Multiporator

®

. The optimal pulse voltage

may be 2 to 3 times higher for suspension cells and 2 to 5 times higher for adherent cells.

Diameter of cell

[µm]

5 530 1100 1100*

Voltage

2-mm cuvette

room temp.

Voltage

4-mm cuvette

room temp.

Voltage

2-mm cuvette

4 °C

Voltage

4-mm cuvette

4 °C

*

10 270 540 540 1100

15 180 360 360 710

20 130 260 260 530

25 110 220 220 430

30 90 180 180 360

35 80 160 160 310

40 70 140 140 270

45 60 120 120 240

50 50 100 100 210

60 40 80 80 160

80 30 60 60 120

* The maximum voltage attainable with the eukaryotic module of the Multiporator

Fig. 4b

48

®

is 1,200 V.

8 Appendix

8.2 Volumes of hypoosmolar and isoosmolar electroporation buffer for setting required osmolarity (10ml)

Desired osmolarity ml hypoosmolar buffer (ml) ml isoosmolar buffer

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

Table 3

8.3 Composition of electroporation buffers

The electroporation buffers supplied by Eppendorf are tested for the following important criteria:

Conductivity

•

•

pH value

•

Osmolarity

•

Sterility
Mycoplasma

•

Endotoxins

•

•

Pyrogens

•

Cytotoxicity

8 Appendix

Poration (hypoosmolar) Poration (isoosmolar)

Sterile bidistilled water Fill up to 1000 ml Fill up to 1000 ml

KCl 25 mM 25 mM

PO

KH

2

4

HPO

K

2

4

myo-Inositol * ad 90 mOsmol/kg ad 280 mOsmol/kg

pH value 7.2 ± 0.1 7.2 ± 0.1

Conductivity at 25 °C 3.5 mS/cm ± 10 % 3.5 mS/cm ± 10 %

* The purity of myo-Inositol may vary greatly from batch to batch. It must be pure enough to ensure that, at

280 mOsmol/kg in bidistilled water, a conductivity of 10 µS/cm is not exceeded. The conductivity of individual
myo-Inositol batches should be measured before the buffer is prepared.

0.3 mM 0.3 mM

0.85 mM 0.85 mM

49

8 Appendix

8.4 Protocol for the electroporation of eukaryotic cells, based on Jurkat

Multiporator

Transfection Protocol

®

8 Appendix

Jurkat

Cell line: Jurkat, T-lymphocyte, human leukemia (suspension cell line)

Transfection with: plasmid pEGFP-N1 (in bidistilled H

Electroporation buffer: Eppendorf hypoosmolar electroporation buffer (PH)

Culture medium: RPMI 1640 / 10 % FCS

Cuvette: Eppendorf, 2-mm gap width, 400 µl

Temperature: RT (20 to 25 °C)

Reference: Prof. Ulrich Zimmermann phone: [phone]

Lehrstuhl für Biotechnologie fax: [phone]
Biozentrum Universität Würzburg e-mail:
Am Hubland, D-97074 Würzburg, [e-mail]
Germany

1. Harvest the cells in the exponential growth phase and centrifuge them

(5 to 10 minutes, 200 x g, room temperature).

2. Resuspend the cells in RPMI 1640 / 0.5 % FCS, determine the number of cells and wash them.

(5 to 10 minutes, 200 x g, room temperature).

O)

2

Note : Incubation time in the electroporation buffer must not exceed 30 minutes to guarantee a successful

electroporation.

3. Resuspend the cells in hypoosmolar electroporation buffer. When doing so, set the cell concentration

to 1 x 10

4. Add and mix plasmid DNA (5 to 20 µg/ml final concentration, in bidistilled H

5. Transfer 400 µl cell suspension into electroporation cuvettes (2-mm gap width).

The cell suspension must be free of air bubbles.

6. Electroporation:

Mode: Eukaryotes " "
Voltage (V) 240 V
Time constant (
No. of pulses (n) 1

7. After the pulse, allow the cell suspension to stand in the cuvette for 5 to 10 minutes at room temperature.

8. Carefully transfer the cell suspension from the cuvette to 3 to 5 ml RPMI 1640 / 10 % FCS and cultivate it in a

60-mm culture dish.

9. Detection methods for transfection :

The expression of the plasmid pEGFP-N1 can be detected clearly after 24 to 48 hours with the aid of FACS analysis
or under a fluorescence microscope.

Result

Survival rate: 70 to 85 %
Transfection rate: 65 to 80 % based on the number of surviving cells .

Results were measured 24 hours after transfection.

6

cells/ml.

O).

2

τ

) 40 µs

55 % based on the initial number of cells used for the experiment .

50

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Tel. [phone]
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Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.es

SWITZERLAND

Vaudaux-Eppendorf AG
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.ch

UNITED KINGDOM

Eppendorf UK Limited
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.co.uk

CANADA

Eppendorf Canada Ltd.
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorfna.com

CHINA

Eppendorf China Ltd.
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.cn

FRANCE

EPPENDORF FRANCE S.A.R.L.
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.fr

JAPAN

Eppendorf Co. Ltd.
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.jp

NORDIC

Eppendorf Nordic Aps
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorf.dk

SOUTH & SOUTHEAST ASIA

Eppendorf Asia Pacific Sdn. Bhd.
Tel. [phone]
Fax [phone]
E-Mail:
asiapacifi[e-mail]
Internet: www.eppendorf.com.my

USA

Eppendorf North America
Tel. [phone]
Fax [phone]
E-Mail: [e-mail]
Internet: www.eppendorfna.com

OTHER COUNTRIES

Internet:
www.eppendorf.com/worldwide

In touch with life

Your local distributor: www.eppendorf.com/worldwide

Eppendorf AG · 22331 Hamburg · Germany · Tel: [phone] · Fax: [phone] · E-Mail: [e-mail]

Eppendorf North America, Inc. · One Cantiague Road · P.O. Box 1019 Westbury, N.Y. 11590-0207 · USA

Tel: [phone] · Toll free phone: [phone] · Fax: [phone] · E-Mail: [e-mail]

Application Support

Europe, International: Tel: [phone] · E-Mail: [e-mail]

North America: Tel: [phone] ext. 2258 · E-Mail: [e-mail]

Asia, Pacific: Tel: [phone] · E-Mail: support_asiapacifi[e-mail]

is a registered trademark.

®

eppendorf

B 4308 900.091-09/0308 · Printed in Germany