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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....

How many stable clones will I get from one transfection?

This depends on the respective cell type (transfection efficiency, viability and integration frequency). If it is important to you to know the frequency of stable integrants before carrying out your actual experiments, the frequency should be...

Do you recommend a purification method to purify specific blood/immune cell populations before Nucleofection®?

We recommend using negative selection (depletion) because your purified cells are "untouched", not influenced, and strictly not activated.Positive selection may cause increasing amounts of dead cells and could also lead to activation of the cells due...

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...

Following Nucleofection®, I cannot obtain stable clones from single cells. What could be the reason?

Some cell lines need certain cell densities to proliferate. For those cells you can try using conditioned medium for setting up limiting dilution or culturing single cells together with feeder cells.

What antibiotic concentration should I use for selecting stable transfectants?

We highly recommend to perform a titration for G418 in a range of 0,1 mg/ml to 1,5 mg/ml; in serum-free culture expand range down to 20 µg/ml).Choose the concentration which is 0,1 or 0,2 mg/ml above the one which shows complete cell death as...

How much DNA should I use in my Nucleofection® Reaction to obtain stable clones?

You should use the amount of DNA recommended in the optimized protocol for transient transfection. If obtaining a very high number of clones is important for your experiments, you can try increasing the amount of DNA. For further tips see our...

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...

My suspension-adapted HEK293 (or CHO) cells tend to clump in culture. How can I avoid this?

Efficient protein production experiments require growth of the host cell in single-cell suspension, something that is sometimes difficult to achieve since most established cell lines retain their adherent growth characteristics. Commercially...

Do bacteria lead to false positive MycoAlert® Results?

NO - the lysis reagent in the MycoAlert® Assay is not strong enough to lyse bacteria / yeast. Also, the enzymes utilized are not very common in bacteria or yeast – but are universal among the mollicute family. Bacterial contamination is...
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