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If I use Miltenyi paramagnetic beads for positive selection of cells, i.e. beads remain on the cell surface, does that influence Nucleofection® results?

No, it seems that the beads bound to the cell surface do not have any influence on Nucleofection®. Larger magnetic beads (i.e. > 1µm) may disturb the Nucleofection® process and negatively influence cell viability and efficiency.

What is the proof that DNA enters the nucleus during Nucleofection®?

There are two facts that prove that the DNA is directly transported into the nucleus: 1.) Using the Nucleofector® Technology, transfection of non-dividing cells is possible. With conventional non-viral transfection methods cell division is a...

How can I remove the cells from the 100 uL Nucleocuvette® Vessel after Nucleofection®? Is there any alternative?

We recommend using the plastic single use pipettes provided in the kits. These pipettes prevent any damage to the cells and ensure a good cell recovery.

Is your pmaxGFP control vector supplied with the Nucleofector® Kits suitable for stable expression?

No. This vector is only supplied in our Nucleofector® Kits as a positive control and cannot be used for selection because it does not contain a mammalian selection marker. The only resistance gene expressed by this vector is kanamycin which is...

What cell number do I need per well when using the 96-well Shuttle® Device or the 4D-Nucleocuvette® Stripes?

The cell number is very much dependent on the cell type you use. For high throughput Nucleofection®, we usually start with one fifth of the optimal amount recommended for the standard Nucleofection® in the 100µl cuvette. Currently the absolute...

What is the amount of DNA needed per well when using the 96-well Shuttle® Device?

The amount depends on the cell type and on the DNA construct. As a rule of thumb 400 ng of DNA per well should be applied. However, for some cells or constructs even 100 ng may be sufficient.

What is the amount of siRNA needed per Nucleofection® Reaction?

When performing siRNA-mediated knockdown experiments it is advisable to conduct a dose-response (concentration) analysis to determine the minimum siRNA concentration necessary for sufficient target knockdown on the mRNA, protein, or functional level....

Do you have any recommended methods for detaching human monocytes from the plate for assaying or before Nucleofection®?

We have had good results by incubating the cells in ice cold PBS for 10 minutes and then rinsing the plates. Alternatively, the cells can be detached without a medium change by gently pipetting the cell suspension up and down. Our experience has also...

Which pipette tips are compatible with the 96-well Shuttle® Nucleocuvette® Plates?

We recommend using special tips, e.g. epT.I.P.S™ (Eppendorf, Hamburg, Germany) or TallTips™ (Matrix Technologies, Hudson, NH, USA). You can contact our Scientific Support Team for a list of compatible tips, including tips for pipetting robots.

How do you recommend that we isolate splenic lymphocytes from mouse spleens for Nucleofection®? Is erythrocyte lysis required?

For the preparation of mouse spleen cells we recommend cutting the spleen once and passing the tissue through a 100µM cell strainer or steel mesh using a plunger. The cells are flushed into a petri dish containing PBS. In order to remove fat, cell...
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