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When nucleofecting cancer cell lines, do I need to be concerned about passage number?

For the most efficient gene transfer, we recommend using cells that are in logarithmic growth phase and at a passage number of 10-15 (from the time of thaw). This is because some cell lines differentiate and change their features after many...

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...

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.

Why am I not able to detect my fluorescent labeled siRNA (e.g. FITC) after 24 or 48 hours post-Nucleofection?

Fluorescently labeled siRNA duplexes can be used to analyze transfection efficiency by fluorescence microscopy or flow cytometry. However, FITC, Rhodamine, or Alexa-488 labeled siRNA oligos should be analyzed 0.5-3 hours post-Nucleofection™. Cy-5...

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.

Do I need a special incubator for cultivation of insect cells ?

Insect cells are cultivated at lower temperatures than E.coli or mammalian cells (S2 cells at 24°C, Sf9 cells at 28°C).Therefore they need a dedicated incubator with appropriate temperature.

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,...

Which promoter can be recommended for transient gene expression in mammalian cells ?

Vectors with a CMV promoter typically work fine for transient protein expression in mammalian cells, like HEK293, CHO or NSO. For insect cells, like Sf9 or Sf21, vectors with insect promoters are required. In HEK293E (EBNA) cells, the use of...

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...

What is the best incubation time for transient gene expression in Nucleofection® Experiments?

The optimal incubation time ranges from 2 to 6 days depending on various factors like the stability of the protein in the medium, potential toxicity of the protein, cell density and stability of the plasmid in the nucleus. To check the timepoint for...