What are the most common causes for low transfection efficiency when using the Neon® device?
Here are some possibilities:
(1) Sub-optimal electrical parameters
(2) Plasmid preparation containing high salt
(3) Plasmid larger than 10 kb
(4) Plasmid concentration too low
(5) Cells are stressed, damaged or contaminated by Mycoplasma
(6) Cell density too low or too high
(7) Cells with high passage number
Answer Id: 5535
What are the most common causes for low cell survival rate using the Neon® device?
Here are some possibilities:
(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
Answer Id: 5536
Will nicked DNA lead to reduced transfection efficiency?
Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid.
The content of “nicked” DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in significant decrease of transfection efficiency.
Answer Id: 5537
Will low A260/A280 ratio of my DNA sample lead to both reduced transfection efficiency and cell viability?
My Neon® system R Buffer has a precipitate. Can I put my bottle in a 37°C water bath to dissolve it?
What are the most important considerations with Neon® system transfection of large plasmids?
For large plasmids, it is important to prepare a highly concentrated solution of the plasmid for electroporation. An example: For the control plasmid, 0.5 ug is used for a 10 ul electroporation. The size of the control plasmid is about 5.5 kb. If your large plasmid is 50 kb, then the size is almost 10 times larger than the control and you have to use 10 times more weight of plasmid to have an equal number of molecules present. So you will need 5 ug of the large plasmid in a 10 ul electroporation to have an equivalent number of plasmids present. In order to keep the volumes low, the plasmid concentration will have to be over 5 mg/ml.
One thing to keep in mind is that when you add high amount of plasmid, it can damage cells due to toxicity of plasmid sample itself. So you will need to optimize the plasmid amount. A recommended strategy would be to start by adding the 50 kb example plasmid up to 2 ug per each 10 ul electroporation. Even though theoretically you should add more than 2 ug due to the large size of plasmid, start with 2 ug to avoid any toxicity. Then check the result. If viability looks OK and efficiency is lower than expected, increase the amount of plasmid in further reactions until you find the optimal amount for best results.
Answer Id: 5540
Does efficiency vary with 10 ul and 100 ul tips in the Neon® System?
There is no reason to speculate that an optimized electroporation parameter would be different between 10 ul and 100 ul tips. Therefore, even though the table in the Neon® cell database states 10 ul tip, those conditions should be OK for 100 ul tip also as long as the density of the cells remains the recommended one. And if the result of 100 ul is much worse than 10 ul, change voltage either increasing or decreasing by 50 to 100V. It should work well with this slight adjustment of voltage.
Answer Id: 5541
Do you have suggestions for improving viability when transfecting HL-60 cells with the Neon® system?
Our online protocol suggests to use 50,000 cells with the 10 microliter tip. However, HL-60 cells do not survive well when transfected at low density. To improve viability you can try using at least 100,000 (better: 200,000) cells with the 10 microliter tips. Also, make sure your plasmid DNA is highly purified, as HL-60 cells are sensitive to LPS. If you observe that viability is good after 24 hours but decreases over the next 72 hours, you may either be using the wrong culture medium (RPMI 1640 + 10% FBS is recommended, do not use DMEM) or your batch of FBS contains low levels of cytotoxic contaminants.
Other possibilities are mycoplasma contamination or very high passage number of your cells. If this is the case please buy a fresh vial of HL-60 cells from ATCC and try your transfections again.
Answer Id: 5543
Can I increase cell viability of Dendritic cells after electroporation?
DNA amount and quality are very critical for electroporation of Dendritic Cells (recommended 0.5-1µg; maximal 2 µg). LPS (lipopolysaccharides) have a strong negative effect on cell viability. Please make sure that all components you use for dendritic cell culture and transfection, e.g. PBS, FCS and especially DNA, are LPS free. We additionally recommend purifying your DNA by precipitating it twice with 20% PEG/2.5 M NaCl. Viability is usually not an issue when working with mRNA or siRNA.
Answer Id: 5544
Do my monocytes or macrophages get activated upon transfection with your Neon® system?
They could get activated, and 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 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 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 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 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 endotoxin-free and do not cause monocyte/macrophage activation in our hands.
Answer Id: 5545
What is your recommended strategy for optimizing siRNA transfection using the Neon® system?
For initial measurement of siRNA uptake, our FITC-labeled BLOCK-iT™ Fluorescent Oligo for electroporation (catalog number: 13750062) can be used. However, the fluorescent signal is somewhat weak and it is not easy to determine whether the oligo has actually entered the cells or is just sticking to the outside of the plasma membrane. In addition the signal fades quickly. Therefore, a better strategy is to use qPCR to measure knockdown of an actual target mRNA such as GAPDH and normalize it to a scrambled negative control.
Answer Id: 5546
What are the benefits of the Neon® Transfection System over standard electroporation?
(1) Small to large number of cells can be used. The transfection is performed using as few as 2 × 10E4 or as many as 8 × 10E6 cells per reaction using a sample volume of 10 ul or 100 ul.
(2) 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 suspension buffers cover all cell types: T buffer (not yet included in the kit) for primary T and B cells, and R buffer for other cells.
(3) Open and transparent protocols that are optimized for ease of use and simplicity. The Neon® Cell Database (www.lifetechnologies.com/neon) contains optimized protocols for many commonly-used cell types.
(4) The Neon® device is preprogrammed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type
Answer Id: 5488
How should I clean the Neon® device and the Pipette station?
Clean 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. If 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
Answer Id: 5489
Is there a difference in how transfection efficiency is measured with the Neon ® System versus the Nucleofector™ Device?
The Neon® system measures the transfection efficiency by the percentage of GFP-positive cells among all cells which include live and dead cells. In contrast, the Nucleofector™ Device measures the transfection efficiency by the percentage of GFP-positive cells among only the live cells.
Answer Id: 5490
How are the Neon® Kit cell viability and transfection efficiency determined by your R&D team?
Cells were analyzed for viability and transfection efficiencies 24 hours or 48 hours post-electroporation using a Guava PCA-96 Cell Analysis System. LIVE/DEAD® cell viability assay populations were calculated by propidium iodide staining (1:2000). The percent of transfected cells was calculated by dividing the number of GFP positive cells by the total population and recorded as transfection efficiencies. The percent of dead cells was calculated by dividing the number of PI-stained cells by the total population
Answer Id: 5491