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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...

Four hours after Nucleofection®, I can see the hepatocytes have attached to the well, but the morphology does not look correct, should I be concerned?

There is no reason for alarm. The hepatocytes may not exhibit normal morphology a few hours after Nucleofection® but by 24 hours post Nucleofection®, morphology should be normal. Remember to perform a fluid change about 4 hours post...

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...

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 kind of protocol you recommend for stimulation of human T cells?

For getting the results we present in our datasheet, we recommend the follwing strategy: Cells should be stimulated and cultivated for 3 days before Nucleofection, preferentially on plates coated with 1 µg/ml anti-CD3 and 2 µg/ml anti-CD28 antibody....

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...

Are there any effects on the differentiation of neural stem cells following Nucleofection®?

The ability of transfected rat or mouse neural stem cells to be subsequently differentiated into a variety of cell types is well documented in:Olig2 Overexpression Induces the In Vitro Differentiation of Neural Stem Cells into Mature...

Can calcium influx issues affect viability or differentiation in Stem Cells after Nucleofection®?

When cells are transfected by Nucleofection®, transient pores are generated in the cell membrane. Generally, these pores disappear within 15 minutes of transfection, but if cells are plated immediately after Nucleofection® in medias that contain...

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...

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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