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.

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

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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.

with microsecond pulse times.

2 Principle of the Multiporator®

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

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

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).

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).

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

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.

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

600

400

200

37%

04080120 160

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

()τ

200

[µs]

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).

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]

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

, pulses with a time constant

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.

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

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

800 µl volume

4°C

020406080100

Cell diameter [µm]

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