Search Results

I currently work with some adherent cell lines, such as 293, which are grown in DMEM media. I have noticed that the mortality seems to be high after Nucleofection®. Do you have any recommendations?

For some adherent cell lines we strongly recommend using RPMI instead of DMEM medium. As described in culture conditions of the cell line optimization kit protocol as well as the NIH3T3 (DSMZ) protocol, using RPMI instead of DMEM as "transfer and...

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...

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 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,...

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...

Data Type

Category

Research Area