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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....

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...

What are the requirements for direct Nucleofection® of mRNA for protein expression?

The mRNA should be capped and polyadenylated. The conditions of Nucleofection® will be the same as for DNA with the particular cell type, i.e. follow the same protocol and use the same program, except one will likely need to add a much higher...

What is the basic principle of the Nucleofector® Technology?

The Nucleofector® Technology is a novel transfection method especially designed for the needs of primary cells and difficult-to-transfect cell lines. It is a non-viral method based on a unique combination of electrical parameters and cell-type...

Why is the Nucleofector®Technology ideal for primary cells and difficult-to-transfect cell lines?

There are several reasons to choose the Nucleofector® Technology for your gene transfer experiments. One is the direct transfer of DNA to the cell nucleus. This makes gene expression independent from cell division. Therefore, the technique is the...

What optimization is necessary to get the Nucleofector® Technology to work?

None. For each cell type in our product list we offer a cell-type specific solution and thoroughly optimized electrical parameters. These are already pre-programmed in the Nucleofector® Device. Additionally our optimized protocols give you...

What can I do when there is no optimized protocol available for my cell of interest?

Lonza is constantly developing new optimized kits and protocols for an increasing number of primary cells and cell lines. In order to obtain the latest information, check our website or contact our Scientific Support Team.We are offering Primary...

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

For Nucleofectio® 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...

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™...
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