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Is the age of the mice important for my mouse T cell Nucleofection®?

Yes. We recommend using mice between 6-12 weeks. Using mouse T cells isolated from younger or older animals for Nucleofection® may result in much lower transfection efficiencies and/or viabilities.

What are the critical steps for successful Nucleofection® of monocytic cell lines like THP-1, HL60 and U-937?

To achieve optimal results, we strongly recommend following the Optimized Protocols in regard to cell culture conditions (medium, splitting cycle, density before Nucleofection), DNA amount and purity.Please also be sure not to exceed 90xg when...

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...

I see activation of my monocytes (or macrophages or DCs) following Nucleofection™. Why is this? What can I do to address this problem?

We have examined the effects of Nucleofection™ (without DNA) on these cells and have not observed significant activation. This indicates that neither the Nucleofector™ Solution, the Nucleofector™ Program nor the recovery medium are sufficient for...

Are there any effects on the differentiation of neural stem cells following Nucleofection™?

The ability of transfected rat or mouse neural stem cells to be subsequently differentiated into a variety of cell types is well documented in: Olig2 Overexpression Induces the In Vitro Differentiation of Neural Stem Cells into Mature...

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...

Can calcium influx issues affect viability or differentiation in Stem Cells after Nucleofection™?

When cells are transfected by Nucleofection™, transient pores are generated in the cell membrane. Generally, these pores disappear within 15 minutes of transfection, but if cells are plated immediately after Nucleofection™ in medias that contain high...

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,...

What are the requirements for direct Nucleofection™ of mRNA for protein expression?

The mRNA should be capped and polyadenylated. The conditions of Nucleofection™ will be the same as for DNA with the particular cell type, i.e. follow the same protocol and use the same program, except one will likely need to add a much higher amount...

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...

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