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Unlike standard cuvette-based electroporation chambers, the Neon NxT Electroporation System leverages the proven Neon technology where electroporation takes place in a biologically compatible pipette tip chamber. The design of a gold-coated wire electrode inside of the 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).

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The optimal time for analysis of protein expression is dependent upon the stability of the protein being expressed. For a short-lived protein, like luciferase, analysis of protein expression 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.

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The benefits of the Neon NxT Electroporation System over other electroporation systems are listed below:

- Small to large number of cells can be used. The transfection is performed using as few as 2 X 10E4 or as many as 10 X 10E6 cells per reaction using a sample volume of 10 µL or 100 µL.
- The Neon NxT Electroporation System uses a single transfection kit (Neon NxT 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. The two NxT resuspension buffers cover all cell types and applications: Neon NxT Resuspension T Buffer for high voltage protocols >1900 V (naive T cells), and Neon NxT Resuspension R Buffer for all other protocols.
- Open and transparent protocols that are optimized for ease of use and simplicity. TransfectionSelect Protocols & Citations Library (thermofisher.com/transfectionprotocolsandcitations) contains optimized protocols for many commonly-used cell types.
- The Neon NxT Device is preprogrammed with 24-well optimization protocols to optimize conditions for all payloads and cell types.
- The Neon NxT Electroporation System leverages Neon technology that produces a more uniform electrical field and a lower pH gradient across the cell suspension. Therefore, this design allows for a better maintenance of physiological conditions resulting in very high cell survival compared to conventional electroporation.

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If you find another cell type in the Neon Nxt Device cell-specific protocols that is similar to your cell type in terms of tissue origin, you can use the electroporation parameters of that cell type for your cell type. You may not get optimal results, but it is a good starting point. For example, say you have 293T cells and you find the HEK293 protocol, since both 293T cells and HEK293 cells are derived from human embryonic kidney, you can use the electroporation parameters of HEK293 cells for 293T cells.

Alternatively, you can use the preprogrammed 24-well optimization protocols in the Neon NxT Electroporation Device to optimize for your cells.

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Yes. The Neon NxT Electroporation System can be used for electroporation of any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type-specific protocol for DNA, or use the pre-programmed 24-well optimization protocols included in the Neon NxT Device.

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We recommend analyzing the cells 72 hours post-electroporation.

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Monocytes/macrophages can get activated upon transfection with the Neon NxT Electroporation System, and there are several possible reasons for this:

Monocytes/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 which has been purified by anion exchange chromatography, such as our PureLink HiPure kits. If you did this and still see monocyte/macrophage activation, perform one or two rounds of PEG precipitation to remove residual endotoxin. Alternatively, you can subject your plasmid to a second round of anion exchange chromatography purification. If you still get monocyte/macrophage activation, the plasmid itself may contain sequences which 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 monocyte/macrophage activation. Please make sure that none of these components activates your cells.

The procedure for isolating your monocytes is also important. We recommend negative selection rather than positive selection, as it leaves the monocytes “untouched” by antibodies.

Our electroporation buffers are endotoxin-free and do not cause monocyte/macrophage activation in our hands.

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The Neon NxT Electroporation System relies on the same unique and trusted electroporation technology as the Neon Transfection System (Cat. No. MPK5000), but it has new features that make it easier to use. The Neon NxT pulse generator has an improved feedback loop with user interface notification, and ClipTip technology has been incorporated into Neon NxT pipette tips for secure attachment and easy ejection, along with other ergonomic design improvements.

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Potential causes for low transfection efficiency with the Neon NxT Electroporation System include the following:
- Sub-optimal electrical parameters
- Plasmid preparation contains high salt concentration
- Plasmid size is larger than 10 kb
- Plasmid concentration is too low
- Cells are stressed, damaged, or contaminated by mycoplasma
- Cell density is too low or too high
- Cells have undergone high number of passages
- Multiple uses of the same Neon NxT tip

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Yes. The Neon NxT Electroporation Device can be UV sterilized inside a bio-safety cabinet.

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The most common causes for low cell survival with the Neon NxT Electroporation Device are listed below:
- Sub-optimal electrical parameters
- Poor plasmid quality such as endotoxin contamination
- Plasmid preparation contains high salt concentration
- Plasmid quantity is too high
- Cells are stressed or damaged
- Multiple uses of the same Neon NxT tip

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Yes, we recommend wiping the Neon NxT Electroporation Device with 70% Ethanol/isopropyl alcohol (IPA) for sanitization.

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Clean the surface of the Neon NxT Electroporation Device and Neon NxT Pipette Station with 70% ethanol/isopropyl alcohol and wipe. Do not use harsh detergents or organic solvents to clean the units. Avoid spilling any liquid inside of the Neon NxT Pipette Station. If you accidentally spill any liquid (e.g., buffer or water) inside the Neon NxT 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 NxT Pipette Station.

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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. We have some preliminary results indicating that very large BAC DNA can be transfected as well, albeit with a low transfection efficiency.

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Yes. However, we recommend waiting for about 4-6 hours before adding antibiotics back to the cells. This is to help ensure that the membrane integrity has been restored.

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