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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...

Is there a difference between, for example, the programs C-20 and C-020?

No, the programs C-20 and C-020 are identical regarding their electrical parameters. The letter minus a two-digit number is the nomenclature of the Nucleofector™ I. Since we have launched the Nucleofector™ II we changed it to a letter minus a three...

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...

Why do you recommend 7AAD instead of PI staining for cell determination in mouse macrophages?

The results obtained using 7AAD staining are much clearer than those obtained using propidium iodide (PI).  Using flow cytrometry stained and unstained, transfected and non-transfected populations can be separated much more easily.

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...

How long have you been able to observe GFP expression in primary Human Keratinocytes?

GFP expression has been observed for up to one week as measured by light and fluorescence microscopy.

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...

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

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

What stock concentration of Propidium Iodide does Lonza use for FACS analysis after Nucleofection®?

We use a stock concentration of 100 µg/ml in PBS. This is diluted 1:300 – 1:400 into the sample.

I've noticed that duration of the Nucleofection® Program seems to change even when I haven't changed the program I am using. Why?

The duration time of a program is dependent on the number of the program. The duration is also dependent on how long the Nucleofector® System has been turned on as well as the number of Nucleofections you have done in the last couple of...
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