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How often should I change the medium during selection of stable clones after Nucleofection®?

The culture medium should be changed every 2-3 days during the selection process.This eliminates potentially toxic substances produced by dying cells and secondly, it keeps the concentration of the antibiotic at a constant level.

Is it more difficult to detect DsRed by FACS than GFP?

Yes, DsRed is much less bright than GFP and is more difficult to detect by FACS and microscopy because it bleaches quickly.

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...

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...

How can I improve Human Hepatocyte viability post Nucleofection on the 96-well Shuttle device?

In addition to proper cell handling and using culture media optimized for long term culturing of hepatocytes (Lonza Basal medium supplemented with BulletKit), we recommend separating the healthy hepatocytes from the dead cells and debris in the...

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 is the best way to centrifuge Human Hepatocytes prior to Nucleofection?

In order to avoid cell compaction in the pellet and difficulties in cell resuspension, we recommend using round bottom 2 ml vials or 50 ml BD Falcon™ tubes for all centrifugation steps prior to Nucleofection™.

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 I use the pmaxGFP control plasmid with insect cells such as S2 cells or SF9 cells?

The pmaxGFP™ Vector provided in our Nucleofector® Kits is not expressed in insect cells. We strongly recommend an insect expression vector encoding a fluorescent protein or lacZ reporter as a positive control for your experiments [e.g. Novagen™’s...
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