How To Optimize Your Electroporation

How to Guide 1 How To Optimize Your Electroporation Carl Robinson, PhD The use of electroporation as a tool to deliver materials into cells was first described in 1972 by Neuman and Rosenheck. The process, which uses short electrical impulses to generate reversible holes in the cell membrane, has subsequently been used to deliver genetic material (DNA and RNA), proteins, drugs or other molecules into mammalian, insect, fungal, plant and bacterial cells. Electroporation has transformed the way in which scientists work. Nevertheless, as with all laboratory techniques, it is not without its fair share of pitfalls and disappointments. Therefore, it is of utmost importance to follow good practices and optimize your experimental conditions to fit your cell type and application. This guide will provide some tips and advice on how to avoid common problems with your electroporation experiments and optimize your protocols and parameters to get the best results. Preparing your samples This one’s just right First, make sure the cells you wish to use are suitable for electroporation, otherwise you will be onto a loser from the start. They need to be healthy, free from mycoplasma or other unwanted infections, free from contaminants and in an appropriate growth phase, typically log phase. When it comes to the electroporation itself, for experimental success, it’s important that cells are kept at the right temperature for the cell type being used – not too hot, not too cold, just right. For many bacterial cells, this means keeping them chilled to slow down their biological processes, reducing harm to the cells during the experiment. However, other cell types, including many mammalian cell lines, do not tolerate chilling well and therefore should be kept warm. It is important to do your homework and find out what is most appropriate for your cells. Finding the sweet spot Check the literature to see if others have made recommendations about the number or ratio of your cells of interest to use if they have performed similar experiments. This can help to give you a starting point from which to work out the number of cells and amount of the target molecule you wish to deliver to achieve optimal efficiency. Initially, this is likely to require a bit of trial and error but remember to do this in a structured way. Use a dilution series of the target with different set amounts of cells to find the sweet spot. Too few cells and the yield of successfully transformed/transfected cells will inevitably be low and cell types that rely on cell-to-cell contact may exhibit poor growth after the experiment. Too high a density of cells and uptake of the target molecule may be inhibited. There isn’t a “right” concentration; you will need to optimize for your particular situation. HOW TO OPTIMIZE YOUR ELECTROPORATION 2 How to Guide Scale to suit Electroporation cuvettes are where the magic happens and they come in a range of sizes to suit differing experimental needs. Consider the amount of material you have available for your experiment – both of cells and insert – and the amount you require at the end of the process and choose accordingly. When it comes to setting up your protocol on the electroporation machine, remember to indicate the correct cuvette size – as the gap distance between the conductive plates will differ – so that it is able to deliver an appropriate impulse for your reaction volume. Salts are an electroporator’s worst friend Salts can be very problematic in electroporation reactions, causing harmful arcing in the machine and consequently cell death. It is highly recommended to wash and resuspend your cells with an appropriate medium to remove excess salts before proceeding. Sucrose is a popular choice for bacterial cells, although specialist electroporation buffers are also a great option and are often used for mammalian cells. This will also help to remove contaminants such as undesirable proteins that can reduce efficiency. You may also need to purify your insert solution too for the same reason, depending on what you have resuspended it in. The ionic strength of your sample buffer will impact its electrical properties so bear this in mind when setting parameters. Selecting your parameters Turning your attention to the electroporation equipment, there are a number of factors to consider. This includes the waveform used, pulse pattern, pulse duration, voltage and capacitance. Many instruments come with several preset protocols, but it’s important to understand what changing each parameter will do. This will help you optimize experiments that are not performing as well as they ought to and will come in useful if there are no preset protocols that suit your need. Conditions may vary from instrument to instrument too, so optimal conditions for one may not hold true for another. Waveform Two waveforms are generally used for electroporation – square waves and exponential decay waves. Square waves rapidly rise to their target voltage, maintain it for their specified duration and then rapidly reduce to nothing again and are popular for mammalian cell electroporation. With exponential decay waves on the other hand, the voltage rises rapidly to the target voltage but then declines gradually over time as a capacitor discharges. This form is more commonly used for bacterial, yeast, plant and insect cells and some mammalian cells. Pulse pattern For some cells, a single pulse is enough to generate usable pores in the cell membrane. However, for others, a series of multiple, lower voltage pulses produce pores more reliably and effectively. The voltage is normally reduced where multiple pulses are used as otherwise permanent damage may be caused to the cells, reducing cell viability. Multiple, lower voltage pulses may also be gentler on fragile cells compared to a single large shock. Pulse length, resistance and capacitance The electrical pulses used in electroporation typically last micro- to milliseconds. For square waves, this is a simple “on and off”, which can be set directly. However, for exponential decay waves, it is a little more complex and is referred to in relation to the time constant (T). HOW TO OPTIMIZE YOUR ELECTROPORATION 3 How to Guide Altering the resistance and capacitance values will determine how long the pulse lasts, as these parameters impact the rate of discharge. When voltage is increased, pulse duration is typically decreased and vice versa to prevent irreparable damage to the cells. As a rule of thumb, pulse times need to be increased where chilled cells are used over room temperature cells to enable pores to be opened effectively in the membrane. Pulse voltage The voltage delivered across the gap between the conductive electrical plates of the electroporation cuvette is known as the field strength and is measured in kV/cm. To get the same pulse, the voltage must be reduced in line with smaller cuvette gap sizes. Cell diameter is also an important consideration when choosing a pulse voltage. Generally speaking, the smaller the cell, the higher the voltage required to successfully create pores. Consider your parameters, particularly if you are performing the electroporation on room temperature cells rather than chilled cells. High voltages and long or multiple pulses can cause the sample to heat up and may reduce cell viability. Ready to go! Dry the cuvette If you are ready to give your selected conditions a test run, there are still more factors to consider. This may sound basic, but it is vitally important that you ensure the cuvette is dry on the outside. This is particularly pertinent when a cuvette has been kept chilling on ice; a thorough wipe down with some lab tissue will suffice. Failure to do so can result in arcing when the electrical pulse is delivered, potentially damaging the equipment and definitely frying your sample! Treat them well A successful electroporation experiment doesn’t end at the “zap”. It’s important to treat your cells well after the event. They’ve just had a shock, quite literally, so will be in a fragile and vulnerable state. As with all living things, they require nurturing back to health, which means providing them with the optimal conditions for growth, including factors such as temperature and nutrition. For some cell types – including many bacterial cells – keeping everything cold was important during the preparation and electroporation steps, but now is the time to warm them up again. Try not to do this too suddenly though, as abrupt changes can result in higher rates of cell death and therefore poorer experimental results. For example, adding cold media to the cold reaction mix and then warming them up gradually in the incubator may be preferable to the sudden shock of bathing your cold cells in warm media. Again, this varies between cell types so find out what is best for your cells. Delays in attending to your cells after electroporation can rapidly reduce viability so make sure you don’t leave them hanging around. The precise conditions to enable the cells to start dividing once more will vary depending upon your target cell type but are likely to be similar to those you use normally to obtain optimal growth. Where selective agents are used, it is generally advisable to refrain from including these immediately following electroporation to allow the cells some time to recover first. For bacteria, this may mean waiting a matter of hours whereas for mammalian cells a day or two’s pause is advisable. HOW TO OPTIMIZE YOUR ELECTROPORATION 4 How to Guide Efficiency is key If you are in the process of optimizing an electroporation protocol, compare the outcomes between the conditions tested. Look at overall cell survival and what percentage of those surviving cells have been transformed/transfected successfully. You should expect to kill a fairly large percentage of your cells. After all, the electrical pulse needs to be strong enough to disrupt the cell membrane, otherwise the pores won’t form, but you need to get enough cells out to make the experiment viable. If the overall cell numbers are low, are you putting enough cells in at the start or are they dying during the electroporation process? If you are getting plenty of viable cells but they do not contain the molecule you are trying to insert then are you adding enough insert to the reaction or are the electroporation conditions suboptimal, leading to poor pore formation or insert entry? Try adjusting parameters and repeating the process to work through the troubleshooting steps, one by one if possible, so you can discern which parameter is responsible for giving the changes you are seeing. It is possible to undertake the electroporation process with a reporter, such as a luciferase, to help with parameter optimization; but this can be less convenient than merely checking cell survival if your lab is not set up for it. One final thing to remember, optimization can be a time-consuming task so only do it if you really need to. If you are performing a one-off experiment and one correct transformant/transfectant is all you need, for example, then consider if it is worth the time investment. Sometimes, just enough is good enough! With all the points discussed above in mind, this guide should give you a head start along the road to electroporation success.