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I have a GFP construct that I use and usually get anywhere from 50-80% GFP positive cells using FACS analysis while I get only 30-40% using a GFP-NFAT fusion protein driven by the same promoter. Do you have any suggestions?

The Amaxa™ Nucleofection™ Conditions are optimised for each cell type, the solutions and programs are cell type dependent and not vector dependant. The expression of the fusion protein depends on the localization of the protein transcription,...

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)...

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

No. The Nucleofector™ Kits are only available as full kits.

Who is the distributor for my country?

Please click on the link provided to search for your distributor.

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...

Can calcium influx issues affect viability of human Mesenchymal Stem Cells (hMSC)?

Yes. When cells are transfected using 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 media that...

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...

What are the critical steps for successful Nucleofection™ of monocytic cell lines like THP-1, HL60 and U937?

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...
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