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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™.

Which reporter gene should I choose for my reporter gene experiment?

In general, all reporter genes can be used. For some applications (e.g. reporter gene experiments in various primary cells, experiments with late analysis time point due to starving and /or stimulation) a reporter gene with a longer half life than...

What is the maximum amount of DNA I can use in my Nucleofection Experiment?

The overall amount of 5 µg should not be exceeded. Look in the cell specific Optimized Protocol (OP) for the recommended total DNA amount of the cell type of interest.

How can I determine the correct centrifugation speed for my particular centrifuge and rotor?

The correct rotor speed can be calculated by measuring the maximum radius of your rotor and entering the information into the table found on the website which can be reached by clicking on the Related Link.

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...

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.

Is the age of the mice important for my mouse T cell Nucleofection?

Yes. We recommend using mice between 6-12 weeks. Using mouse T cells isolated from younger or older animals for Nucleofection™ may result in much lower transfection efficiencies and/or viabilities.

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...

Does the recovery medium contained in the Mouse T Cell Nucleofector Kits contain any immunogenic substances?

No. The post Nucleofection™ Recovery media contains no immunogenic factors and should not influence cell stimulation or differentiation. For Mouse T cells, some experiments have shown that after Nucleofection™ cells can be stimulated efficiently in...

I see activation of my monocytes (or macrophages or DCs) following Nucleofection. Why is this? What can I do to address this problem?

We have examined the effects of Nucleofection™ (without DNA) on these cells and have not observed significant activation. This indicates that neither the Nucleofector™ Solution, the Nucleofector™ Program nor the recovery medium are sufficient for...
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