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Are rat and mouse hepatocytes transfected by Nucleofection® still functional?

Yes, transfected mouse hepatocytes show albumin synthesis rates comparable to those of untransfected cells. Moreover the morphology and polarization of rat hepatocytes is not affected by Nucleofection®.

Can I freeze the 96-well Nucleocuvette® Plates?

Yes. Freezing of the plates at -20°C does not alter their properties.

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...

Can I integrate the 96-well Shuttle® Device into a liquid handling system?

Yes. The 96-well Shuttle® Device is designed in such a way that it is addressable by robot grippers. Furthermore, the software contains the required interface to be connected with the LHS software. However, the practical implementation for each...

After Nucleofection®, how do you determine cell viability?

We determine cell viability after Nucleofection® in two ways: 1) FACS determination of viable/dead cells by PROPIDIUM IODIDE STAINING. We normally analyze transfection efficiency in living cells by FACS: We first exclude cellular debris by...

What is the optimal size of DNA that you recommend for Nucleofection®?

We routinely use plasmids of 4-7 kb in our laboratories and plasmids up to approximately 20kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Some preliminary results we have also...

Generation of a stable cell line: A positive control plasmid works, but I cannot obtain stable clones with my construct of interest. What could be the reason?

One reason could be that your gene product is toxic to the cells. We recommend testing the construct for expression of the gene of interest by transient Nucleofection® of your cells. If the proportion of expressing cells drops between 4 and 48 hours...

Can I use the Nucleofector® 2b Solutions and Optimized Protocols with the 4D-Nucleofector® X-Unit or the 96-well Shuttle® System?

No. The 4D-Nucleofector and 96-well Shuttle® System uses specifically optimized electrical parameters and solutions. Five different 4D-Nucleofector or 96-well Shuttle® Solutions are available for primary cells. For cell lines, three...

How long does it take to obtain stable transfectants?

Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition.

How should I purify my DNA for Nucleofection® of neurons?

The quality and the concentration of DNA used for Nucleofection® in general plays a central role for the efficiency of gene transfer. We strongly recommend using endotoxin free prepared DNA. Endotoxin free Kits are available from several...
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