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Why do you have different Optimized Protocols for the Nucleofection® of unstimulated and stimulated Human T cells?

We found that the stimulation of T cells not only changes the cells' function but also significantly alters the requirements for successful Nucleofection®. That's why Lonza developed two Optimized Protocols for transfection giving different...

Is it possible to use frozen primary CD34+ hematopoietic stem cells for Nucleofection®?

Yes. For cryopreserved PBMCs or enriched CD34 populations, we recommend culturing the thawed cells 1-2 hours in culture medium before Nucleofection®. Any further enrichment procedure after thawing is not recommended. Viability of frozen nucleofected...

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...

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...

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...

Do you have a protocol for the Nucleofection® of Plasmodium berghei using the Nucleofector® Systems

Nucleofector I/II/2bWe have excellent data for the Nucleofection™ of Plasmodium berghei from the group Chris J. Janse and Andrew P. Waters from the University of Leiden. Their protocol describes a transfection efficiency of 10-3 – 10-4. With this...

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...

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