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Is it possible to use different substrates (e.g. siRNA duplexes) with the 96-well Shuttle™ Device?

Yes. Transfection can be done with any substrate AND under exactly the same conditions. For example, the same conditions (96-well Shuttle™ Solution and program) are used for siRNA transfection or shRNA vectors. This greatly facilitates the...

What is the best way to establish the Nucleofector™ Technology in my lab?

We strongly recommend establishing the Nucleofector™ Technology with the positive control vector pmaxGFP™ provided in our kits. pmaxGFP encodes the green fluorescent protein (GFP) from Copepod Pontellina sp. Just like eGFP expressing cells, maxGFP™...

Is there a special recommendation on Human T cell enrichment before Nucleofection™?

For Nucleofection™ it is preferable to use Human T cell populations enriched by magnetic separation. For example, MACS™ Microbeads (Miltenyi) or by a rosetting method. For magnetic separation we recommend negative selection or detaching beads after...

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 only...

Are there any recommendations to avoid excessive mortality during T cell Nucleofection™?

Try to keep the DNA amount for Nucleofection™ quite low (e.g. 1 µg plasmid DNA per 100 µl reaction) since higher DNA amounts might cause increasing toxicity and mortality. It is also important to use highly purified DNA, e.g. by using Endofree™...

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...

How can I increase cell viability of Dendritic cells after Nucleofection™?

DNA amount and quality are very critical for Nucleofection™ of Dendritic Cells (recommended 0.5-1 µg; maximal 2 µg). LPS (lipopolysaccharides) have a strong negative effect on cell viability. Please make sure that all components you use for dendritic...

I want to stimulate Human T cells post Nucleofection™. Are there any precautions to keep in mind?

After Nucleofection™, wait at least four hours before stimulation. Stimulating immediately after Nucleofection™ may lead to increased cell mortality and poor activation of cells. For more details on the stimulation of Human T cells post...

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...

Is it possible to get neomycin resistant clones after Nucleofection™ with pmaxGFP™?

The pmaxGFP control DNA is not suitable for making stable clones because it lacks a selectable marker for mammalian cells. The kanamycin resistance expressed by this plasmid is only suitable for bacterial selection.

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