Electroporation Support—Getting Started

Here are the special features of the Neon® transfection System:

1. The Neon® Transfection System uses a single transfection kit (Neon® Kit) that is compatible with various mammalian cell types, including primary and stem cells, thereby avoiding the need to determine an optimal buffer for each cell type. Two cell resuspension buffers are provided to cover all cell types: T buffer for primary T and B cells, PBMCs, monocytes, and bone marrow–derived cells, and R buffer for established adherent and suspension cells as well as primary adherent cells.

2. Small to large numbers of cells can be used in the Neon® Transfection System. The electroporation is performed using as few as 1 × 104 or as many as 5 × 106 cells per reaction using a sample volume of 10 μL or 100 μL.

3. Open and transparent protocols are available, that are optimized for ease of use and simplicity. The Neon® Cell Database contains optimized protocols for many commonly used cell types.

4. The Neon® device is pre-programmed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type.

The Neon® Transfection system is the second generation of the Microporator (MP-100) from Digital Bio/NanoEntek. The unique feature of both these instruments is that they use an electrode pipette tip as a transfection chamber instead of a standard electroporation cuvette. The performance of these instruments is comparable, but there are some differences. The main difference is in the user interface. The Neon® Transfection System unit has updated firmware and can upload more programs than the Digital Bio Microporator MP-100. The MP-100 Microporator is white in color and has a smaller screen than the Neon® Transfection System unit. The Neon® Transfection Pipette Station is not compatible with the MP-100 pipette (white in color). This is because the sensor connector (it is a 2-pin connection) is different from that in the Neon® Pipette Station (12-pin connection). We do not carry an adapter to allow compatibility. The kits and components in both systems are exactly identical except that they are branded as Neon® in the Neon® Transfection System. The old manual can be used for the Neon® Transfection System instrument. More information regarding the history of the Neon® Transfection System can be found here.

Unlike standard cuvette-based electroporation chambers, the Neon® system uses a patented biologically compatible pipette tip chamber. The design of a gold-coated wire electrode inside a pipette tip has been shown to produce a more uniform electrical field and a lower pH gradient across the cell suspension. Therefore, this design allows for better maintenance of physiological conditions, resulting in very high cell survival compared to conventional electroporation (Kim JA, Cho K, Shin MS, et al. (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23(9):1353–1360).

The Neon® Transfection System efficiently delivers nucleic acids (DNA/siRNA) into all mammalian cell types. In comparison, lipid-based transfection reagents are less efficient for delivering nucleic acids into hard-to-transfect cells like primary cells, stem cells, and hematopoietic cells. Viral delivery such as those using lentivirus and adenovirus can efficiently deliver DNA and siRNA from our shRNA or miRNA vectors into difficult-to-transfect cells, but producing the virus is a time-consuming and more complex process.

The Resuspension Buffer (Buffer R or T) is used to resuspend the cells prior to electroporation, whereas the Electrolytic Buffer (Buffer E or E2) is used for electroporation and is added to the Neon® tube prior to electroporation. 

Both R and T Resuspension Buffers are used to resuspend cells prior to electroporation. Resuspension Buffer R is the standard cell resuspension buffer that is used to resuspend established adherent and suspension cells as well as primary adherent cells. Resuspension Buffer T is an alternative cell resuspension buffer that is used to resuspend primary T and B cells, PBMCs, monocytes, and bone marrow–derived cells. Its composition differs from that of Buffer R and allows the application of higher voltages due to lower conductivity. It does not work with established cell lines or primary cells, which have been kept in culture for some time. In situations where it is not immediately clear whether Buffer R or Buffer T would work, we recommend testing both in separate optimization experiments.

We recommend using T buffer instead of the standard R buffer for primary blood-derived suspension cells such as primary T and B cells, PBMCs, monocytes, and bone marrow–derived cells. These cells are smaller than regular cell types and require higher voltage for successful electroporation. If you use R buffer and apply high voltage (over 1800 V), you will see sparks or arcing, regardless of cell number and other conditions. The maximum voltage for R buffer is around 1900 V. Buffer T composition differs from that of Buffer R to allow the application of higher voltages due to lower conductivity.

We do not offer Resuspension Buffer R as a stand-alone item. The kits contain buffer reagent in excess to use the Neon® tips up to 2 times. 

The E2 buffer has higher osmolarity than E buffer. Higher osmolarity prevents the leakage of electroporation content from the 100 µL Neon® tip, which has a larger hole at the tip end than the 10 µL Neon® tip (pore diameter of the Neon® tips: 100 µL tip = 2.10 mm; 10 µL tip = 0.65 mm).

All buffer compositions are proprietary.

The Neon® device uses a square pulse wave; the pulse width ranges from 1–100 ms and the pulse number ranges from 1–10. Volts range from 500–2500 V.

The interval between pulses is fixed at 1 ms.

Yes, the Neon® tip does fit with Digital Bio/NanoEntech’s pipette.

The shelf life of the Neon® kits is 1 year from the date of purchase.

No. The Neon® kits are only available as full kits for up to 50 or 192 reactions. The Neon® transfection tubes are available separately (Cat. No. MPT100). The Neon® kits contain sufficient buffers to perform 2 electroporations per tip.  

The Neon® Pipette is permanently calibrated by the manufacturer and does not require any further calibration.

We recommend cleaning the surface of the Neon® device and Neon® Pipette Station with a damp cloth. Do not use harsh detergents or organic solvents to clean the unit. Avoid spilling any liquid inside of the Neon® Pipette Station. In case you accidentally spill any liquid (e.g., buffer, water, coffee) inside the Neon® Pipette Station, disconnect the station from the main device and wipe the station using dry laboratory paper. Invert and leave the station for 24 hours at room temperature for complete drying. Do not use an oven to dry the Neon® Pipette Station.

For each cell type in our Neon® Cell Database, we offer thoroughly optimized electroporation parameters along with a universal electrolytic buffer. These conditions may have to be modified slightly for your particular cell line, since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions listed should be understood as a starting point for your own optimization. For cell lines that are not listed in our database, there is a pre-programmed optimization protocol built into the Neon® device.

Our control plasmid is a 5.9 kb pCDNA™6.2–based expression construct expressing EmGFP from a CMV promoter. The purification procedure is proprietary, but the purity of this plasmid is equivalent to two rounds of anion-exchange chromatography. This plasmid is not meant to be propagated, and we cannot confirm any selectable markers.

Cell fusion may occur "accidentally" as a side-effect during transfection using the Neon® system, for some cell-types that tend to form cell clusters (e.g.. PC-12 cells), but unfortunately, we do not offer a Neon® program that is optimized for cell fusion applications.

Currently, we do not offer a Neon® protocol for electroporation of bacterial cells. However, the Neon® Transfection System may be used for electroporation of Chlamydomonas. Please refer to page 13 in the GeneArt® Chlamydomonas Engineering Kit manual and page 18 in the GeneArt® Chlamydomonas TOPO® Engineering Kit manual for electroporation recommendations.

We currently do not have a validated protocol. However, we could suggest some parameters to try based on a recent publication (Alvarez-Erviti et al., Nature Biotech 29(4):341–347, 2011), where cells were electroporated with a non-identified cuvette-based electroporator. The parameters used were 400 V and 127 mF. For the Neon® system, this translates to about 1350 V. We would recommend running the optimization protocol with this voltage in mind; however, it is difficult to recommend pulse width or number of pulses—thus, optimization is necessary. 

We recommend using anion-exchange chromatography to prepare transfection-grade plasmid DNA. This technology is found in our PureLink® HiPure line of plasmid purification kits. For large plasmids (>50 kb), do not use the PureLink® HiPure purification kits that contain filters or precipitators to avoid damage to your plasmid. We do not recommend using spin columns for plasmid purification, as they contain silica membranes that do not remove impurities to the same extent as anion-exchange resins.

We do not have in-house data, but several reports from customers using a variety of cell lines suggest that it works.

Circular and linearized plasmids that do not contain special recombination sequences transfect with the same efficiency and integrate into the genome with similar probability. However, the area of recombination on the plasmid can be influenced by linearization, as loose ends are preferred over continuous stretches of sequence. By linearizing the plasmid, you can determine the position within the plasmid where the recombination occurs, thereby conserving the expression cassette in most cases.

In general, electroporation is a size-dependent transfection technique and transfection efficiency declines as plasmid size increases. We routinely use plasmids of 4–7 kb in our laboratories, and plasmids up to approximately 20 kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Preliminary results indicate that bacterial artificial chromosomes (BACs) can be transfected as well, but with a low transfection efficiency. Keep in mind that in terms of molarity, 1 mg of a 6 kb plasmid corresponds to 2 mg of a 12 kb plasmid. This has to be factored in when comparing transfection efficiencies of plasmids of different sizes. As an example: when comparing the transfection efficiency of 1 mg of a 10 kb plasmid to the transfection efficiency of a 150 kb BAC, 15 mg of the BAC would have to be used. This is not feasible since DNA amounts that large will have toxic effects on the cells. On the other hand, this does not mean that BACs cannot be transfected using the Neon® system. However, transfection efficiencies with a large amount of DNA will be very low. 

The toxic effects that are seen with some large plasmids are not related to their size but are likely due to sequences located on the plasmid, contaminants in the plasmid preparation such as LPS, or very large amounts being used for electroporation. Always use anion-exchange chromatography–based kits (such as our PureLink® HiPure kits) to prepare transfection-grade plasmid DNA and avoid overloading the columns, as this will result in plasmid preparations of low purity.

Application of a strong electrical field weakens the cell membrane and induces pore formation, which allows the antibiotics to enter the cells. Cell death is induced most likely via toxic metabolic intermediates. In addition, streptomycin has been shown to bind to eukaryotic ribosomes and may directly interfere with protein translation. If your cells need to be cultured in the presence of antibiotics, you can add them back a few hours after electroporation.

• If you find another cell type in the Neon® Cell Database that is similar to your cell type in terms of tissue origin, you can use the parameters of that cell type for your cell type. This does not guarantee that you will get the best results, but it is a good starting point. For example, if you have 293 T cells and you find a protocol for HEK293 cells in the Neon® Cell Database, you can use the electroporation parameters of HEK293 cells for 293 T cells, since both are derived from human embryonic kidney.
• You can use the pre-programmed 24-well optimization protocol in the Neon® device to optimize conditions for your cell type.
• Contact Technical Support for further discussion.

These settings were selected based on our experience optimizing numerous cell lines and primary cells in-house. If none of these settings transfect plasmid DNA into your cells, it is unlikely that other conditions will. However, if low transfection efficiencies are obtained with some of the settings, it is likely that they can be further increased by performing additional optimizations to fine-tune your parameters for voltage, pulse width, and number of pulses.

We do not offer such a service at this time. The Neon® Transfection System is designed to facilitate the optimization of transfection conditions. Typically, three rounds of optimization are sufficient to find the best instrument settings for any given cell line or primary cell type. Unless you prepare your cells from little amounts of tissue or tissue that is difficult to process, optimizing the conditions should not take more than a week and would cost a lot less than a custom service would.

The Neon® Transfection Tubes are disposable and we recommend using each tube for a maximum of 10 times for the same plasmid/siRNA/cell type, to minimize the possibility of cross-contamination. In addition, we strongly recommend that a new Neon® tube be used for a different plasmid DNA/siRNA or cell type, to avoid cross-contamination. If you need extra Neon® tubes to accommodate your experiment, they can be purchased separately (Cat. No. MPT100).

We strongly advise against washing the Neon® tips. Washing will not remove DNA or siRNA attached to the tip and will increase the risk of cross-contaminating the samples. Also, the tips cannot be sterilized, easily increasing the risk of microbial contamination of cultures.

To avoid contamination caused by carry-over from one sample to another, we recommend that you do not re-use the Neon® tip. However, if you are performing a 24-well optimization or if you are performing your transfections in duplicate, you may use tips 2 times. The reason for this recommendation is that the wire electrode inside the tip is coated with 24-karat gold, and some of the gold is released each time an electrical pulse is delivered. Therefore, repeated use of the tip will result in a thinning of the gold coating, causing the conductivity of the tip to change. We found that this effect becomes measurable after three uses. If you want to be absolutely sure that the correct voltage and current are delivered to your cells, use the tip only twice.

If using the same plasmid/siRNA and the same cell type, one can use the Electrolytic Buffer for up to 10 times and then change the tube and buffer together. If a different plasmid/siRNA or cell type is used, we recommend changing the buffer after two usages to avoid carryover contamination.

Yes. The Neon® Transfection System can be used for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type–specific protocol for DNA or pre-programmed 24-step optimization protocol.

The siRNA concentration in Neon® transfection refers to the siRNA concentration in the culture medium and not to the siRNA concentration in the electroporation content in the Neon® tip. For example, if electroporation is performed with the 100 µL Neon® tip and the transfected cells are plated in a 24-well plate that contains 500 µL culture medium, the siRNA concentration is measured as the concentration in the 500 µL culture medium and not the concentration in the 100 µL electroporation content.

A good start is to use the plasmid electroporation parameters for the same cell type. The Neon® Cell Database contains optimized plasmid electroporation parameters for many commonly used cell types. If the Neon® Cell Database does not contain your cell type of interest, you can use the 24-well optimization protocol that is pre-loaded on the Neon® device. Please contact Technical Support if you should need further assistance.

In most cases, instrument settings that were optimized for a certain cell line or primary cell type using a plasmid will also work with siRNA. However, those settings may not be the most optimal ones for the delivery of siRNA. Therefore, additional optimization may be required to improve knockdown efficiency of the target. For cell lines or primary cell types that have not been optimized with plasmid DNA, a 24-well optimization is the best approach to find optimal conditions. Keep in mind that for every condition tested, a negative control siRNA needs to be transfected in order to normalize knockdown efficiency.

The Neon® Cell Database contains optimized protocols for many commonly used cell types. However, these conditions may have to be modified slightly for your particular cell line, since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions listed should be understood as a starting point for your own optimization. For cell lines that are not listed in our database, there is a pre-programmed optimization protocol built into the Neon® device.

We currently do not have data to support this, but co-transfection of different plasmids should work. However, the amount of DNA should be carefully titrated, since overloading the cells with plasmid DNA or using unfavorable ratios of the plasmids may cause toxicity. Therefore, we recommend starting optimizations of co-transfection experiments with low amounts of DNA followed by a stepwise increase. Various ratios of the plasmids should be tested if toxicity is observed.

Yes, we have in-house data that show knockdown of EmGFP by a specific siRNA that was co-transfected with an EmGFP-expressing plasmid, as well as knockdown of endogenous genes.

In-house, we have transfected as few as 30,000 cells using the 10 µL tips. If the cell density is too low during electroporation, viability will typically be compromised. This effect is somewhat cell type–dependent. Therefore, how low you can go with your cell line or primary cell type without substantially reducing viability needs to be determined empirically. One of our customers has reported successful transfection of 5000 primary hair follicle cells.

According to Neon® guidelines, you can run up to 8 million cells in 100 µL. If the cells are small enough, up to 10 million cells can be used in 100 µL.

After electroporation, wait for about 4–6 hours before adding antibiotics back to the cells. This is to make sure that the membrane integrity has been restored.

Because electroporation uses electric shock to make pores on the cell membrane, this can damage certain membrane proteins. But those membrane proteins will be recovered as time goes by. The recovery time may vary and will depend on the type of cell and protein. There is no general guideline for this yet. In primary cells, which do not proliferate after electroporation, this membrane damage can be permanent so that it hinders certain membrane proteins.  

There is no reason to speculate that an optimized electroporation parameter would be different between 10 µL and 100 µL Neon® tips. Therefore, even though the table in the Neon® Cell Database specifies the 10 µL tip, those conditions should be okay for 100 µL tips, as long as the recommended density of cells is used. If reduced transfection efficiency is observed, fine-tuned adjustment of the voltage settings may be required to improve efficiency. A 24-well optimization with the 100 µL tips is usually not necessary.

The optimal time point for analysis of protein expression is related to the stability of the protein being expressed. The half-life of protein products can range from less than a few minutes to several days. For a short-lived protein (like luciferase), protein expression analysis should be done at 6–18 hours post-electroporation. For a more stable protein such as GFP, the analysis can be done 24 hours post-electroporation or even a little later.

As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA half-life can range from minutes to days (Ross J, 1995, Microbiol Rev 59:423–450) while the half-life of protein products can range from less than a few minutes to several days. In general, the recommended time course ranges from 12 to 72 hours to knock down target mRNA and 24 to 96 hours to adequately knock down target proteins. We recommend measuring mRNA knockdown by qPCR at 8, 24, 48, 72, and 96 hours post-electroporation to determine the time point for maximum knockdown. Also, perform time-course analysis to determine protein knockdown by ELISA (more accurate) or immunoblotting (less accurate).

Cell viability is the number of cells that are confirmed viable from a total cell population. Transfection efficiency is the number of cells that are successfully expressing your construct out of the total number of viable cells (i.e., GFP-positive cells). 

Cell viability can be determined by staining cells with propidium iodide or by the trypan blue exclusion method. For adherent cells, cell detachment can be performed using Trypsin or TrypLE™ Express enzyme prior to staining. Transfection efficiency can be determined using a fluorescence microscope with filter settings appropriate for the detection of GFP (emission: 509 nm). Cells may be counted wither by FACS or using the Countess® Automated Cell Counter. 

To determine the Neon® transfection efficiency for siRNA, we recommend transfecting the cells with a fluorescent-labeled negative control siRNA (BLOCK-iT™ Fluorescent Oligo, Cat. No. 13750062) and measuring the transfection efficiency by the percentage of fluorescent-stained cells among viable cells. However, keep in mind that there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescence detection with a microscope does not distinguish the siRNA that enters the cell from the siRNA that sticks to the cell membrane. To measure transfection efficiency more accurately, one needs to transfect the cells with a positive control siRNA such as the one that targets a house-keeping gene, and measure the knockdown of target RNA or protein.