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Do the Clonetics Rat and Mouse Astrocytes require an extracellular matrix for plating?

No, they simply need to be seeded onto tissue culture treated plastic. For best performance, we recommend to use positively charged plasticware

What does the message "weak" mean on the display of the Nucleofector® IIN, IIS or 2b after Nucleofection®?

This message means that the system cut off the pulse before it was completed (usually this is because the conductivity of the solution is at the low end of the normal range). The pulse was applied; it is just going to be a little weaker than...

What age are the rats that your Clonetics Rat DRGs are isolated from?

The Rat Neonatal Dorsal Root Ganglion (DRG) Neurons (R-DRG-505) are isolated from neonatal, about 2-3 days old.The Rat Embryonic Dorsal Root Ganglion (eDRG) Neurons (R-eDRG-515) are isolated from embryonic rats (E14-15).

Generation of a stable cell line: A positive control plasmid works, but I cannot obtain stable clones with my construct of interest. What could be the reason?

One reason could be that your gene product is toxic to the cells. We recommend testing the construct for expression of the gene of interest by transient Nucleofection® of your cells. If the proportion of expressing cells drops between 4 and 48 hours...

How long does it take to obtain stable transfectants?

Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition.

Will the GFAP expression in your Clonetics Normal Human Astrocytes remain the same throughout the culture?

The GFAP expression decreases rapidly with passaging. The results that we give are performed in the 1st passage out of cryopreservation.

Where can I find the meaning of the 4D-Nucleofector® System Error Codes?

In the back of the respective manuals, there is a chart giving a brief description of the error codes. If more information is needed or an error code is recurrent, it is best to contact Scientific Support for advice to determine if there is a...

In stable cell line generation, I have a very high transfection efficiency, but most of my cells die during selection. Is this to be expected?

This is the normal pattern you should expect to see. Only a small proportion (1/10,000 to 1/100) of all transfected cells will integrate the transfected DNA into their genome and become stable transfectants. The remaining cells lose the transfected...

What medium and which cytokines are recommended for Natural Killer (NK) cell culture?

For the cultivation of Natural Killer (NK) cells we recommend using the X-VIVO 15 medium.In regard to cytokines, this varies depending upon the application, but many cited publications indicate using IL-2 for activation and longer term culture.

What selection markers can I use for the generation of stable cell lines?

The most commonly used marker is the neomycin phosphotransferase (neo) gene that confers resistance to G418 to eukaryotic cells. Other markers are Puromycin, Hygromycin, Zeocin, or the HPRT gene that can be used in HPRT-deficient cells.
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