What is the advantage of your Neon™ transfection chamber design over standard cuvettes?

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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).

Answer Id: E9080

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Would Neon™ system transfection affect functional study of membrane proteins?

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As electroporation uses electric shock to make pores on cell membrane, this can damage certain membrane proteins. But those membrane proteins will generally be re-expressed and recover 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.

An exception are primary cells which do not proliferate after electroporation, where the membrane damage could be permanent such that it hinders certain membrane protein studies. However, this is quite rare. Given sufficient time to recover, for most proliferating cells there should be no problem with membrane proteins.

Answer Id: E5532

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What are the common causes for low cell survival rate using the Neon™ device?

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Here are possible causes for low transfection efficiency using the Neon™ device:

1. Sub-optimal electrical parameters
2.Poor plasmid quality such as endotoxin contamination
3 .Plasmid preparation containing high salt
4. Plasmid quantity too high
5. Cells are stressed or damaged
6. Multiple uses of the same Neon™ tip
7. Microbubbles in tip, causing arcing

Answer Id: E9130

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How does the Neon™ Transfection System compare to other delivery methods (i.e., lipid-based transfection, viral delivery)?

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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.

Answer Id: E9081

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Can the Neon™ System be used for co-transfecting siRNA and a plasmid construct?

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We have data which shows knockdown of EmGFP by a specific siRNA which was co-transfected with an EmGFP expressing plasmid, as well as knockdown of endogenous genes.

Answer Id: E5520

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Why do my monocytes/macrophages get activated upon transfection with your Neon™ system?

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There are several possible reasons for this. Monocytes and macrophages respond to very low levels of endotoxin (LPS), which could have been introduced with your plasmid DNA. Make sure that you use plasmid DNA that has been purified by anion-exchange chromatography, such as our PureLink™ HiPure Plasmid Purification Kits. If you still observe activation, you may subject your plasmid to a second round of anion-exchange chromatography purification. If you still get activation, the plasmid itself may contain sequences that stimulate the production of Interferon gamma. It is also possible that certain components in your culture medium, including the FBS batch you are using, may cause activation. Please make sure that none of these components activates your cells. The procedure for isolating your monocytes is also important. We recommend negative rather than positive selection, as it leaves the monocytes “untouched” by antibodies. Our electroporation buffers are guaranteed to be endotoxin-free and do not cause monocyte/macrophage activation in our hands.

Answer Id: E8994

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After electroporation, how long should I wait before analyzing protein expression?

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The optimal time for protein analysis is related to the stability of the protein being expressed. For a short-lived protein, like luciferase, analysis should be done at 6-18 hours after electroporation. For a more stable protein, such as GFP, you should start the analysis at 24 hours or longer post-electroporation.

Answer Id: E5533

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My Neon™ system R Buffer has a precipitate. Can I put my bottle in a 37°C water bath to dissolve it?

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No, the precipitate that forms in this buffer is irreversible.

Answer Id: E5539

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What is the difference between the Neon™ Resuspension and Electrolytic Buffers?

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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.

Answer Id: E9082

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What kind of optimization do I need to do to get the Neon™ Transfection System to work?

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For each cell type in our Neon™ Cell Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html), 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.

Answer Id: E9095

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What is the minimum number of cells you can transfect in one reaction with the Neon™ System?

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We have transfected as few as 30,000 cells using the 10 microliter 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.

Answer Id: E5521

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How should I determine the cell viability and transfection efficiency with the Neon™ Transfection System?

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Answer

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 either by FACS or using the Countess™ Automated Cell Counter.

Answer Id: E9127

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Where can I find Neon™ instrument settings for my cell line?

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Go to our "Learn More about the Neon™ Transfection System" page at www.thermofisher.com/neon. There you will find a link to our cell transfection database which currently contains in-house optimized settings for over 140 cell lines and primary cells. 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 which we have posted on our website should be understood as a starting point for your own optimization.

Answer Id: E5516

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Can you provide some tips for determining siRNA transfection efficiency with the Neon™ Transfection System?

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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 one that targets a housekeeping gene, and measure the knockdown of target RNA or protein.

Answer Id: E8995

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What optimization is necessary to get the Neon™ System to work?

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For each cell type in our cell database, we offer thoroughly optimized electrical parameters along with a universal electrolytic buffer. Very little optimization is required. For cells that are not listed in our database, there is a pre-programmed optimization protocol built in the Neon™ device.

Answer Id: E5509

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