efficient and gentle electroporation of eukaryotic cells
Eppendorf Multiporator ~ overview
The basic unit of the Multiporator is made up of a module designed to electroporate eukaryotic cells.
Two optional function modules extend the application range of the Multiporator to either cell fusion or the transformation of bacteria and yeasts. No additional devices or external parts are necessary.
Together with the buffers, cuvettes and chambers that are precisely suited for the corresponding applications, the Multiporator forms the perfect system.
Electroporation of eukaryotic cells
The Multiporator in combination with the specially designed electroporation buffers is optimally balanced for efficient and gentle electroporation of eukaryotic cells.
Soft Pulse technology applies extremely short electric pulses for the highest survival rates. Cell-damaging influences, such as changes in pH values, aluminum release and electrophoresis of the cell content, are minimized. The relevant parameters of voltage and pulse duration are directly set, and the patented electronic pulse discharge ensures that they will be maintained exactlyâ€”independent of the sample resistanceâ€”for reliable and reproducible results.
The hypoosmolar buffer system enables the cell to swell up, thus enabling easier membrane penetration which leads to high transfection rates. The ion composition is adapted to the inner cell environment and stabilizes the Na+/K+ gradient across the cell membrane.
Applications include the transfection of animal and human cell lines, embryonic stem cells, primary cells, oocytes and plant cells.
Recent experiments have proven that the Multiporator is specially suited for the transfer of siRNA into cell lines and primary cells.*1, 2, 3
*1 Sergio Gonzalez, Daniela Castanotto, Haitang Li, Simon Olivares, Michael C. Jensen, Stephen J. Forman, John J. Rossi, Laurence J.N. Cooper: Amplification of RNAi-Targeting HLA mRNAs (Molecular Therapy, Vol. 11, 2005)
*2 Reena P. Vishwanath, Christine E. Brown, Jamie R. Wagner, Hunsar B. Meechoovet, Araceli Naranjo, Christine L. Wright, Simon Olivares, Dajun Qian, Laurence J.N. Cooper, Michael C. Jensen: A quantitative high-throughput chemotaxis assay using bioluminescent reporter cells (Journal of Immunological Methods, 2005)
*3 Lindsay M. Shafer and Lee W. Slice: Anisomycin induces COX-2 mRNA expression through p38MAPK and CREB independent of small GTPases in intestinal epithelial cells, (Biochimica et Biophysica Acta (BBA) â€“ Molecular Cell Research, 2005)
Eppendorf Multiporator ~ features
Electroporation for the transient or stable transfection of eukaryotic cells
Electroporation of bacteria, yeasts, and other microorganisms
Electrofusion of mammalian cells, plant cells and oocytes
Multiporator product features
Compact unit - small and portable
Robust housing with disinfectable surfaces
Extremely simple operation and clearly arranged display
Directly adjustable voltage and time constant
Patented electronics* ensure accurate performance of the pulse parameters, independent of the sample resistance
Soft Pulse™ Technology prevents any negative effect on cells (changes in pH values, the release of aluminum, electrophoresis of the cell content)
An innovative, hypoosmolar buffer system boosts transfection efficiency for eukaryotes
Microprocessor-controlled pulse discharge*1
Optimised buffer system
Outstanding safety levels provided by the built-in cuvette chamber
CE, UL, and CAS approval
2 years warranty
Connectors for external electrodes
Upgradeable with optional functional modules
Electrofusion buffer system sterile, pyrogen- and endotoxin-free, mycoplasm-tested
Device with insert for cell fusion and Helix chamber
Helix chamber High-precision cuvette with two platinum wires wound in parallel and a 250 Âµl capacity Microfusion chamber for the optimisation of cell alignment and fusion parameters under microscopic control
Eppendorf Multiporator ~ Technical Specifications
Area of application:
Electroporation of animal and human cell
lines, primary cells, plant cells, and oocytes
15500 µs, in 5 µs increments
199, with 1 min. time interval
Aluminum, sterile; gap width: 1 mm, 2
mm, 4 mm
Hypo- and isoosmolar electroporation
mycoplasm- and pyrogen-free
Area of application:
Electroporation of gram-positive and
gram-negative bacteria, and yeast
5 ms (nominal)
Electronic safety switch for eliminating
Area of application:
Electrofusion of mammalian cells, plant
cells, and oocytes
15500 µs, in increments of
199, in time intervals of 1 sec.
symmetrically to 0 V
2 MHz sinus
030 sec. before and after pulses
Eppendorf Multiporator ~ ordering
We have endeavoured to provide Australian catalogue numbers; however it would be prudent to verify any catalogue numbers taken straight from our site with POCD Scientific before using them to place an order.
for eukaryotic cells
for eukaryotic cells, bacteria and yeasts
for eukaryotic cells and cell fusion, with 1 Helix fusion chamber and 1 Micro fusion chamber
for eukaryotic cells, bacteria, yeasts and cell fusion, with 1 Helix fusion chamber and 1 Micro fusion chamber
What devices does Eppendorf supply for electroporation?
1. The Electroporator 2510 qand the new Eppendorf Eporator are especially suitable for porating bacteria and yeasts. The time constant permanently set at 5 ms allows new protocols to be optimized simply by selecting a pulse voltage between 200 and 2,500 V.
2. In combination with the electroporation buffers, the Multiporator makes an ideal system for transfecting eukaryotic cells. In combination with the hypoosmolar buffer, the Âµs pulse (Soft Pulse) which handles cells gently efficiently transfects cells at a simultaneously high survival rate. The microprocessor-controlled pulse discharge facilitates optimization by directly programming the time constant and thus guaranteeing the reproducibility of experiments.
The Multiporator is available or can be upgraded with two optional modules. The bacteria module can be used to perform electroporation on bacteria and yeasts. Equipping the module for cell fusion involves a Micro fusion chamber to optimize parameters and a Helix fusion chamber to perform the real fusion experiment.
What quantity of sample can be placed in the Helix fusion chamber?
The Helix fusion chamber can be filled with 250 Âµl of sample.
How should the Micro fusion chamber be cleaned?
The chamber should be cleaned with distilled water before the cells have dried out. Heavier contamination can also be removed using a soft toothbrush or a cleaning agent (e.g. Edisonite Super, order no. 60210, VWR). It is possible to sterilize the chamber by treating it with ethanol (70 %, non-denatured), but the Micro fusion chamber is an open system and normally serves only to optimize parameters.
To what maximum pulse count can the Multiporator be programmed?
The maximum number is 99, with an interval of 60 seconds in each case.
In which solution purified DNA should be soluted in order to use it for electroporation?
DNA should be soluted in very pure water. Compared with TE buffer, the results using aqueous solutions of DNA are substantially better. Especially EDTA, even in Âµmol concentrations, is strongly toxic as a complexing agent in the cell. Both electroporation buffers from Eppendorf can also be used to dissolve DNA, however these buffers should not be used for the elution of DNA in nucleic acid purifying kits.
How should the Helix fusion chamber be cleaned?
The beaker and the core of the Helix fusion chamber should be rinsed thoroughly with distilled water immediately after the experiment to prevent drying of sample residues. If it is severely contaminated, the Helix fusion chamber can be cleaned briefly in an ultrasound bath (possibly with a cleaning additive like Edisonite-Super, for example) or with a very soft (tooth) brush. If using a brush for cleaning, you must ensure that you brush in the direction of the winding, otherwise the electrodes may be moved making the Helix fusion chamber unusable.
Sterilization can be effected by treating with ethanol (70 %, non-denatured). To this end, the beaker is filled with 250 Âµl of ethanol and the core screwed into the beaker. After 10 seconds, the core is unscrewed and the alcohol can be removed. For subsequent drying, place the beakers and the core in the stand under sterile conditions (e.g. clean bench).
The electrodes of standard electroporation cuvettes are made of aluminium. Is there a danger that cytotoxic aluminium ions are released during the pulse and cause damage to the cells ?
"Pulses in the millisecond range intensify the hydrolysis of water, causing a steep pH gradient to form between the electrodes. Under acidic conditions, particularly aluminium is dissolved. The resulting high concentration of aluminium can have a very detrimental effect on the survival rate. With the microsecond pulses of the Multiporator, this effect no longer occurs.
Literature: Friedrich et al, Bioelectrochemistry and Bioenergetics 47 (1998), 103-111"
How can bacteria be transformed successfully using ligation preparations?
Ligation preparations generally include reaction buffers containing salts to increase the conductivity of the sample. Since this may affect the electrical parameters of the device and thereby can lead to a lower transformation rate, the conductivity of the ligation preparation should be reduced by one of the standard methods below.
1. Precipitate the ligated DNA by adding ethanol or butanol and glycogen as described in Biotechniques 16, 988.
2. Dilute the ligation preparation with water.
Is it possible to change the pulse duration of the Multiporator or is it fixed?
The pulse duration (time constant) of the eukaryotic module can be freely selected between 15 and 500 Âµs in 5 Âµs increments. The pulse duration of the bacteria module is permanently set to 5 ms.
How is it possible to check which osmolarity of electroporation buffer is still tolerable for a cell line?
For this purpose, the cells are incubated in different mix ratios of hypoosmolar and isoosmolar buffer for 30 min. Starting from the hypoosmolar buffer, osmolarity should be increased in increments of approx. 50 mOsmol. The survival rate of the cells is then examined (e.g. using Trypan blue staining). Taking account of the fact that at least 90 % of the cells in the buffer must survive, the buffer with the lowest possible osmolarity should be selected. A mixing table is included in the Basic Application Manual for electroporation, for example.
Is it necessary following the electroporation to incubate eukaryotic cells on ice for 10 minutes, as is frequently recommended in the protocols?
As a rule it is not necessary to cool down the cells with ice following the electroporation. At 4 Â°C the "resealing" of the membranes is delayed, so that over a longer period of time an exchange of molecules takes place between the cell and its environment. This causes increased stress and can reduce the survival rate. When the closing of the membranes is to be slowed down intentionally e.g. to introduce large molecules), the cells should be cooled down with ice for a maximum of two minutes and then immediately transferred to a temperature of 37 Â°C.
Can PBS or medium be used as the electroporation buffer with the Multiporator?
No, this is not recommended. Since the conductivity of the PBS or cell culture medium is relatively high, this would result in too high a current flow. This would cause massive damage to the cells. The Multiporator is optimized for buffers with a low conductivity, therefore Eppendorf buffers should be used for the electroporation of eukaryotic animal cell.
Can the protocols for the Electroporator 2510 (bacteria and yeasts) also be used for the Multiporator?
These protocols cannot be performed on the basic Multiporator model (eukaryotic module). However, they can be used with the optional bacteria module of the Multiporator.
Are the microsecond pulses of the Multiporator sufficient to rupture the membrane ?
Yes. Rupture of the membrane already takes place after about 15 Âµs. The remaining time is needed to widen the "pores". Pulses in the millisecond range can cause massive damage to cells, e.g. due to electrophoresis.
What is the composition of the Eppendorf buffers used for electroporation and electrofusion?
The buffer components are listed in the relevant application manual for electroporation/electrofusion.
What is the maximum number of eukaryotic cells which can be used for an electroporation?
We recommend using a cell concentration of 1 x 106 cells/ml. In this range, the electric field can still efficiently influence the cells. At higher cell densities (>3 x 106) homogeneous field conditions are no longer ensured, i.e. the cells are no longer subjected to a uniform electric field. This can lead e.g. to a decrease in the transfection rate. Using a cell density < 1 x 106 cells/ml should have no effect on the transfection rate.
What is the procedure for establishing a new protocol for the electroporation of eukaryotic cells?
1. Check whether the cells will tolerate the hypoosmolar buffer. After 30 minutes' incubation, 90 % of the cells should survive, otherwise mixtures of hypoosmolar and isoosmolar buffer will have to be prepared until the desired survival rate is achieved.
2. Cell size can be used as a guide to the voltage to be used. A good approach is to make a rising series of voltages in 50 V increments. The time constant is usually 40 - 100 Âµs. Detailled information are included in the "General Protocol" and "Optimization Protocol" or in the Basic Application Manual for Electroporation which can be found here.
Can the Multiporators be upgraded with the module for bacteria or the electrofusion module?
Yes, all Multiporators can be upgraded with the respective upgrade kits. Please note that the module has to be installed by a service technician.
Can external electrodes be connected to the Multiporator?
Yes, this is possible via the "insert for connecting external electrodes" (order no. 4308 021.004). Modification of the Multiporator is not required for this. The external electrodes can be used both for electroporation experiments (all Multiporator models) and for electrofusion (devices with a fusion module). The insert has a function switch to adapt it to the method selected. The external electrodes are connected to the insert via 4 mm all-insulated lab plugs.
Can the resistance and/or the capacity of the Multiporator be set?
No, these values cannot be set in any of the Multiporator modules. The advantage of the Multiporator is the fact that the time constant which results from capacity and resistance can be input directly and this results in the microprocessor-controlled pulse.
Why is it advantageous to use the Eppendorf hypoosmolar electroporation buffer for the transfection of animal cells with the Multiporator?
In combination with the device, the buffers form a perfect system for electroporation experiments. They not only have low conductivity and an ion composition adapted to the inner cell environment; the hypoosmolar buffer in combination with Soft Pulse also facilitates gentle transfection. Under hypoosmolar conditions the cells swell as a result of water uptake. Because of the increasingly large size of the cell and the loosened cytoskeleton, electroporation can take place at a lower voltage. Furthermore, the cells assume a uniform spherical shape, which enables an effective optimization of the pulse parameters. A test of the cells for compatibility with the buffer must be performed beforehand. In addition, it is necessary to ensure that the cells are not subjected to hypoosmolar conditions for more than 30 minutes.