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When should I begin looking for the Luciferase signal after Nucleofection™?

The time course of plasmid expression after Nucleofection™ might be different from the time course seen with other transfection methods. We recommend looking at different time points and start with the first time point as early as 4 to 6 hours post...

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,...

What should I pay attention to when transferring my reporter gene set up from a cell line to a primary cell?

In general the overall expression level differs between different cell types. Don't expect the same level of gene induction when working with primary cells. You might need to change to other DNA amounts or ratios.

Does your Nucleofector™ Solutions contain anything that would inhibit attachment of adherent cells?

No. In many cases where decreased attachment is a problem, the cause is inactivated trypsin. Unless the trypsin is inactivated with trypsin inhibitor or media containg BSA or serum, it will continue to degrade the cells and ultimately decrease cell...

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...

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...

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 PI. Using flowcytrometry stained and unstained, transfected and non-transfected populations can be separated much more easily.

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 gating for...

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.

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...

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