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When nucleofecting cancer cell lines, do I need to be concerned about passage number?

For the most efficient gene transfer, we recommend using cells that are in logarithmic growth phase and at a passage number of 10-15 (from the time of thaw). This is because some cell lines differentiate and change their features after many...

Why am I not able to detect my fluorescent labeled siRNA (e.g. FITC) after 24 or 48 hours post-Nucleofection?

Fluorescently labeled siRNA duplexes can be used to analyze transfection efficiency by fluorescence microscopy or flow cytometry. However, FITC, Rhodamine, or Alexa-488 labeled siRNA oligos should be analyzed 0.5-3 hours post-Nucleofection™. Cy-5...

I have been having trouble with "clumping" with my PC12 cells. Do you have any recommendations?

Yes. As PC12 cells often tend to clump we recommend resuspending these cells as thoroughly as possible with a sterile 1mL pipette tip or transfer pipette from your Nucleofector™ kit. This circumvents potential fusion of cells due to the...

What is a Neural Stem Cell (NSC)?

Neural stem cells are adult stem cells but you also find them in embryos (already in "adult" differentiation status) and they have the potential to develop into neurons and glial cells. We recommend isolating them from rat and mouse embryos because...

Do I need a pure neuronal culture for Nucleofection™ ?

In principle, it should be absolutely fine to nucleofect pure neuron cultures (e.g. after purification by Ficoll). Glial cells are not necessary for transfection. What one would have to consider for example, is to get the required number of neurons,...

Does your Nucleofector™ Solutions contain anything that would inhibit attachment of adherent cells?

No. In many cases where decreased attachment is a problem, the cause is inactivated trypsin. Unless the trypsin is inactivated with trypsin inhibitor or media containg BSA or serum, it will continue to degrade the cells and ultimately decrease cell...

What are your recommendations for minimum and maximum cell numbers for Nucleofection™ ?

The recommended cell number will vary depending on which Optimized protocol is being used. In general, using less than 2x10exp5cells per reaction causes a major increase in cell mortality. For some cell lines we have tried cell numbers up to...

After Nucleofection™, how do you determine cell viability?

We determine cell viability after Nucleofection™ in two ways: 1) FACS determination of viable/dead cells by PROPIDIUM IODIDE STAINING. We normally analyze transfection efficiency in living cells by FACS: We first exclude cellular debris by gating for...

What is the optimal size of DNA that you recommend for Nucleofection™?

We routinely use plasmids of 4-7 kb in our laboratories and plasmids up to approximately 20kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Some preliminary results we have also...

How should I purify my DNA for Nucleofection™ of neurons?

The quality and the concentration of DNA used for Nucleofection™ in general plays a central role for the efficiency of gene transfer. We strongly recommend using endotoxin free prepared DNA. Endotoxin free Kits are available from several suppliers....

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