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How can I remove the cells from the 100 uL Nucleocuvette® Vessel after Nucleofection®? Is there any alternative?

We recommend using the plastic single use pipettes provided in the kits. These pipettes prevent any damage to the cells and ensure a good cell recovery.

Can I use the Nucleofector® Technology for RNAi applications? How do I start?

Yes. The Nucleofector® Technology can be easily applied for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type specific Optimized Protocol for DNA vectors (cDNA, shRNA or miRNA expressing plasmids) or...

Can I order any of the Nucleofector® Kit components separately?

No, we do not offer the components of the Nucleofector® Kits separately. The Nucleofection® Kits are only available as complete kits.

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 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...

Is your pmaxGFP control vector supplied with the Nucleofector® Kits suitable for stable expression?

No. This vector is only supplied in our Nucleofector® Kits as a positive control and cannot be used for selection because it does not contain a mammalian selection marker. The only resistance gene expressed by this vector is kanamycin which is...

Do you have any recommended methods for detaching human monocytes from the plate for assaying or before Nucleofection®?

We have had good results by incubating the cells in ice cold PBS for 10 minutes and then rinsing the plates. Alternatively, the cells can be detached without a medium change by gently pipetting the cell suspension up and down. Our experience has also...

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...

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