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What can I do when there is no optimized protocol available for my cell of interest?

Lonza is constantly developing new optimized kits and protocols for an increasing number of primary cells and cell lines. In order to obtain the latest information, check our website or contact our Scientific Support Team.We are offering Primary...

Is there a special recommendation on Human T cell enrichment before Nucleofection®?

For Nucleofectio® it is preferable to use Human T cell populations enriched by magnetic separation. For example, MACS™ Microbeads (Miltenyi) or by a rosetting method. For magnetic separation we recommend negative selection or detaching beads...

Are there any recommendations to avoid excessive mortality during T cell Nucleofection®?

Try to keep the DNA amount for Nucleofection® quite low (e.g. 1 µg plasmid DNA per 100 µl reaction) since higher DNA amounts might cause increasing toxicity and mortality. It is also important to use highly purified DNA, e.g. by using Endofree™...

How can I increase cell viability of Dendritic cells after Nucleofection®?

DNA amount and quality are very critical for Nucleofection® of Dendritic Cells (recommended 0.5-1 µg; maximal 2 µg). LPS (lipopolysaccharides) have a strong negative effect on cell viability. Please make sure that all components you use for dendritic...

I want to stimulate Human T cells post Nucleofection®. Are there any precautions to keep in mind?

After Nucleofection®, wait at least four hours before stimulation. Stimulating immediately after Nucleofection® may lead to increased cell mortality and poor activation of cells. For more details on the stimulation of Human T cells post...

Are your Nucleofector® Solutions checked for RNase activity during your QC process?

Yes. We do test the Solutions with a real time RT-PCR based assay. This assay basically measures the quantity of an RNA template after incubation in our solution. In case there is RNAse contamination the template would be digested which would be...

My Nucleofector® Solution was frozen. Is this a problem?

As long as the supplement was not added to the Nucleofector® Solution, then there is no risk of any damage to the solutions. Even long-term storage of several months did not alter the performance of the Nucleofector® Solution. However, if the...

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...

What kind of buffer do you recommend for resuspending my siRNA?

Please use the buffer that is recommended by your siRNA manufacturer.

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...
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