Frequently Asked Questions | Electroporation

Ingenio® Electroporation Kits and Solution

Ingenio® Solution is a broad-spectrum electroporation solution that is compatible with most conventional electroporation instruments including Bio-Rad® Gene Pulser, Harvard-BTX® electroporators, and Lonza-Amaxa® Nucleofector® devices. Many physiologically relevant cell types are refractory to existing chemical transfection methods of nucleic acid delivery. Electrotransfection using Ingenio® Electroporation Kits and Solution achieves high efficiency delivery of plasmid DNA and siRNA into hard-to-transfect cell lines and primary cells. Ingenio® Solution provides a low toxicity alternative compared to other electroporation buffers such as PBS and Bio-Rad® GenePulser® Electroporation Buffer.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the Ingenio® Solution?
Q2. Will the Ingenio® Kits and Solution work for electroporating any target cell type?
Q3. Will the Ingenio® Kits and Solution work with my electroporation instrument?
Q4. What range of delivery efficiency can I expect with my cell type when using the Ingenio® Kits and Solution?
Q5. What sort of cellular toxicity is seen when electroporating using Ingenio® Solution?
Q6. Can I use the Ingenio® Kits and Solution to deliver plasmid DNA? siRNA/microRNA? Large RNA?
Q7. Can I use the Ingenio® Kits and Solution to deliver protein?
Q8. Mirus offers several different Ingenio® Kits. Which kit should I use?
Q9. Lonza-Amaxa® offers several different solutions for use with the Nucleofector® Devices. How can the Ingenio® Solution compare to all of these?
Q10. Can I reuse the Ingenio® Cuvettes?
Q11. Are there any publications using Ingenio®?

PROTOCOL QUESTIONS AND ANSWERS

Q12. What is the recommended final cell density for electroporation using Ingenio® Kits and Solution?
Q13. What pulse type or wave form should I use for my electroporations?
Q14. How do I determine the exact pulse conditions for electroporating my target cell type?
Q15. How much DNA should I include in my electroporation reaction?
Q16. How much siRNA should I include in my electroporation reaction?
Q17. Will antibiotics in the culture medium affect electroporation results?
Q18. Do I need to keep the Ingenio® cuvettes on ice prior to electroporation?

TROUBLESHOOTING QUESTIONS

Q19. Is the passage number of my cells an important consideration for electroporation?
Q20. I observe arcing when performing electroporation. What should I do?
Q21. What can I do to improve electroporation efficiency when using Ingenio® Electroporation Kits and Solution?
Q22. What can I do to reduce cellular toxicity post-electroporation?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of Ingenio® Solution?
The exact composition of the Ingenio® Solution is proprietary. However, it does not contain any animal-derived components.

Q2. Will Ingenio® Kits and Solution work for electroporating any target cell type?
Electroporation is a high efficiency method of nucleic acid delivery to a wide variety of cell types. Ingenio® Electroporation Solution is a universal solution that has been shown to work with a wide variety of mammalian cell types including hard to transfect cell lines and primary cells. For additional information, please consult the Reagent Agent® transfection database or user protocol (PDF).

Q3. Will Ingenio® Kits and Solution work with my electroporation instrument?
Ingenio® Solution has been validated for use with many electroporation instruments, including Bio-Rad® Gene Pulser Xcell™, Harvard Apparatus BTX® ECM, and Lonza-Amaxa® Nucleofector® II devices. Ingenio® Cuvettes are compatible with Bio-Rad® Gene Pulser Xcell™, Harvard Apparatus BTX® ECM, and the Lonza-Amaxa® Nucleofector® I and II devices. Ingenio® cuvettes are similar to conventional electroporation cuvettes provided for mammalian cell electroporation by other manufacturers. However, Ingenio® cuvettes are not compatible with the Lonza-Amaxa® 4D-Nucleofector® System.

Q4. What range of delivery efficiency can I expect with my cell type when using Ingenio® Kits and Solution?
Every cell type responds differently to electric pulses. High electroporation efficiencies can be achieved by optimizing the following key parameters: nucleic acid concentration, pulse type and pulse conditions. Electroporation efficiency data measured with GFP flow cytometry is available for a variety of cell types on our Ingenio® product web page. If you are using Ingenio® Kits and Solution with the recommended settings on the Lonza-Amaxa® Nucleofector® I or II Device, you should observe transfection efficiencies similar to the Nucleofector® kits. See Lonza-Amaxa® comparison data.

Q5. What sort of cellular toxicity is seen when electroporating with Ingenio® Solution?
The physical process of electroporation and the associated use of high voltage pulses generally results in significantly more cellular toxicity compared to chemical transfection. However, use of Ingenio® Solution yields higher cell viability compared to other electroporation solutions such as PBS or Bio-Rad®’s Gene Pulser Electroporation Buffer. If you are using the Lonza-Amaxa® Nucleofector® Device, you can expect to observe cell viability comparable to that achieved using the Nucleofector® kits, when measured twenty-four hours post-electroporation.

Q6. Can I use Ingenio® Kits and Solution to deliver plasmid DNA, siRNA/microRNA, or large RNA?
Yes, Ingenio® Kits and Solution will deliver any nucleic acid. Mirus Bio scientists have electroporated DNA, siRNA and microRNA into various cell types. Additionally, we have received feedback from researchers reporting successful electroporation of large RNA using Ingenio® Electroporation Solution.

Q7. Can I use Ingenio® Kits and Solution to deliver protein?
While this application has not been tested at Mirus Bio, electroporation has been used for delivering protein inside mammalian cells.

Q8. Mirus Bio offers several different Ingenio® Kits. Which kit should I use?
The Ingenio® Kits include either 0.2 cm (blue cap) or 0.4 cm (red cap) cuvettes. You should choose a kit depending on the cuvette size and the electroporation instrument available. The 0.2 cm cuvettes hold 100 µl per electroporation reaction and can be used with most electroporation instruments. The 0.4 cm cuvettes hold 250 µl per reaction and can be used with most conventional electroporators except the Lonza Nucleofector® I and II Devices.

Q9. Lonza-Amaxa® offers many different solutions for use with Nucleofector® Devices. Why do Ingenio® Kits only contain one solution?
Ingenio® Solution is a broad-spectrum electroporation solution that works well with many different cell types and provides similar results to the various solutions provided in Nucleofector® kits when used with the recommended program setting. There is no need to purchase multiple solutions for electroporating different cell types, providing a cost-effective alternative.

Q10. Can I reuse Ingenio® Cuvettes?
Mirus does not recommend reusing Ingenio® Cuvettes due to increased likelihood of contamination and diminished performance. Residual cell debris or detergent can alter the electric pulse, leading to suboptimal electroporation.

Q11. Are there any publications citing Ingenio®?
Yes, the most recent Ingenio® citations can be found in the Citations Database under Technical Resources.

PROTOCOL QUESTIONS AND ANSWERS

Q12. What is the recommended final cell density for electroporation using Ingenio® Kits and Solution?
The optimal cell density for electroporation can vary depending upon the cell type. It is best to empirically determine the optimal cell density for your cell type and conditions. Generally, the optimal cell density falls in the range from 1-10 × 10⁶ cells/ml.

• For suspension cells such as HL-60, Jurkat, K562, etc., we generally recommend higher cell densities close to 1 × 10⁷ cells/ml.
• For adherent cell types such as primary cortical neurons, near-diploid immortalized keratinocytes, etc., we recommend a final cell density in the range of 1-5 × 10⁶ cells/ml.

For specific recommendations, consult the Reagent Agent® transfection database.

Q13. What pulse type or wave form should I use for my electroporations?
Exponential decay and square wave forms are the most commonly used pulse types for mammalian cell electroporation, and Ingenio® Electroporation Solution can be used with both types. If you are just beginning to optimize the electroporation settings for your cells on a conventional electroporator such as a Bio-Rad® Gene Pulser or Harvard-BTX® instrument, we recommend beginning with an exponential decay pulse. Detailed instructions on how to electroporate your target cell type can be found in the Ingenio® user protocol (PDF).

Q14. How do I determine the exact pulse conditions for electroporating my target cell type?
Mirus provides detailed pulse conditions for many cell types that have been electroporated in our labs using different instruments and pulse types. Please refer to the user protocol (PDF) for starting recommendations for exponential-decay or square-wave pulse type. When using Ingenio® Electroporation Solution with Lonza-Amaxa® Nucleofector®, use the suggested program settings for your target cell type by Lonza-Amaxa®. For specific recommendations, consult the Reagent Agent® transfection database.

Q15. How much DNA should I include in my electroporation reaction?
We suggest using a final concentration of 20 µg DNA/ml of electroporation volume. When using a 0.2 cm cuvette, we recommend using 2 µg total DNA in the Ingenio® cell suspension. When using a 0.4 cm cuvette, 4 µg total DNA can be used in the Ingenio® cell suspension. Further optimization can be performed by trying DNA concentrations in the range of 5–50 µg/ml of final electroporation volume.

Q16. How much siRNA should I include in my electroporation reaction?
We recommend a final concentration of 250-750 nM siRNA in the electroporation reaction.

Q17. Will antibiotics in the culture medium affect electroporation results?
No. Antibiotics can be used in the culture medium used to grow the cells; they will not affect the efficiency when used in the cell culture medium before or after electroporation. However, we recommend that you do NOT add antibiotics to the Ingenio® Electroporation Solution during the electroporation. High antibiotic influx during electroporation can cause cellular toxicity.

Q18. Do I need to keep the Ingenio® cuvettes on ice prior to electroporation?
No. Electroporations should be carried out at room temperature.

TROUBLESHOOTING QUESTIONS

Q19. Is the passage number of my cells an important consideration for electroporation?
Yes. A very high or low cell passage number can reduce electroporation efficiency. Use a similar passage number among experiments to ensure reproducibility.

Q20. I observe arcing during electroporation with Ingenio®. What should I do?
Arcing usually occurs when the electroporation sample is too conductive. There are several reasons that a sample will arc, including:

1. DNA dissolved in a high salt buffer
2. DNA concentration >100 µg/ml in the electroporation mix
3. Very high cell density
4. Elevated degree of cell lysis
5. Reuse of cuvettes

Troubleshoot arcing:

• DNA has been prepared using an ion exchange column with a final ethanol precipitation step, exchange the DNA solution to a salt-free or low salt solution.
• If arcing occurs, dilute the DNA and repeat the electroporation. If arcing continues, perform an electroporation without DNA to test whether the cells are contributing to arcing.

Q21. What can I do to improve electroporation efficiency when using Ingenio® Electroporation Kits and Solution?
Low electroporation efficiency could be due to several different factors. Try the following troubleshooting tips for optimizing your electroporation results. For additional details, please refer to the Ingenio® user protocol (PDF).

1. Cell density too low – Determine the optimal cell density for each cell type.
2. Cells not in active growth phase – Divide the cells 18-24 hours before electroporation.
3. Low-quality DNA (partially degraded or contaminated with inhibitor such as endotoxin) – Use highly purified, sterile, contaminant-free DNA for electroporation. Do not use DNA prepared using miniprep kits or procedures. We recommend using MiraCLEAN® Endotoxin Removal Kit (MIR 5900) for removal of endotoxin from your DNA preparation. Alternatively, use cesium chloride gradient or anion exchange purified DNA to limit endotoxin levels.
4. Post-electroporation incubation time not optimal – Determine the best incubation time for each cell type and experiment.
   • For protein expression, the time between electroporation and the peak expression of target protein will depend upon many factors such as the half-life of the expressed protein or type of nucleic acid used (DNA or RNA). Perform a time course experiment from 12-72 hours to determine the peak
     expression period. The optimal incubation time is generally 12-48 hours.
   • For knockdown experiments, a broader time course (12-96 hours) may be required depending upon the half life of the target mRNA and/or protein. The optimal incubation time is generally 24-72 hours.
5. Infection with mycoplasma – Mycoplasma contamination can alter electroporation efficiency and cannot be detected visually. Check your cells for mycoplasma contamination using standard techniques, for example, PCR based detection. Use a new frozen stock of cells or treat your cell culture with appropriate antibiotics to eliminate mycoplasma.

Q22. What can I do to reduce cellular toxicity post-electroporation?
The high membrane permeability caused by electroporation may lead to increased cellular toxicity. Listed below are some suggestions to improve cell health post-electroporation. For additional details, please refer to the Ingenio® user protocol (PDF).

1. Electroporation pulse strength may be too high – Decrease the voltage by increments of 10 V and/or decrease the capacitance by increments of 100 µF.
2. DNA preparation has too much salt – Elute the DNA solution in a low salt solution or buffer exchange against a salt-free or low salt solution.
3. Excessive amounts of DNA in the electroporation reaction – Reduce the amount of DNA used for electroporation. Final DNA concentrations as low as 5 µg DNA/ml may be used for electroporation.
4. Low-quality DNA (partially degraded or contaminated with inhibitor such as endotoxin) – Use highly purified, sterile, contaminant-free DNA for electroporation. Do not use DNA prepared using miniprep kits or procedures. We recommend using MiraCLEAN® Endotoxin Removal Kit (MIR 5900) for removal of endotoxin from your DNA preparation. Alternatively, use cesium chloride gradient or anion exchange purified DNA that limit endotoxin levels.
5. Cells not transferred to complete growth media in a timely manner – Do not allow cells to incubate in Ingenio® Electroporation Solution for more than 30 minutes post electroporation.
6. Expressed target gene is toxic to the cells or an essential gene is knocked down upon siRNA electroporation – Include appropriate controls to assess the effects of target gene expression or knockdown. Try reducing the amount of DNA in case of target gene expression being toxic.