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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...

Can I use larger cuvettes for my Nucleofection Reaction ? Can I use the cuvettes more than once?

No. The electrical parameters provided by the Nucleofector™ Device are optimized for the cuvettes contained in the Nucleofector™ Kits. The cuvettes are single-use only. Using the cuvettes more than once will result in higher cell death and lower...

Do you recommend positive selection or depletion for the purification of Human monocytes for Nucleofection®?

We only recommend the depletion method. For Example, the RosetteSep™ Isolation Kit for human monocytes [Stem Cell Technologies, Cat. No 15028]. The advantage of depletion is that the monocytes are left untouched by antibodies during the process.

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.

Does it matter if the PBS used for monocyte enrichment contains calcium and magnesium, if the cells should be used later in Nucleofection®?

The PBS should be calcium and magnesium free to prevent clumping of cells. We routinely use PBS without calcium and magnesium for example Lonza Cat. No. BEBP17-516Q

What is the molecular weight (MW) of your control plasmid pmaxGFP?

The molecular weight is approximately 2.3x10e6 g/mol based on the average MW of a base pair being 660 g/mol.

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 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.
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