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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.

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™.

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...

When can I start induction studies on Human Hepatocytes transfected with the Nucleofector?

We recommend allowing the transfected hepatocytes to recover in Plating medium for 72 hours post-Nucleofection™. For induction studies (e.g. CYP3A4), the Plating medium is replaced with maintenance medium (William's E without FCS and EGF) and...

What is the lowest amount of Human Hepatocytes that can be used with the 96-well Shuttle System or the 4D-Nucleofector X-Unit?

Human hepatocytes do not like to be cultured and transfected at low densities. Therefore, using less than 100,000 cells per Nucleofection™ is not recommended.

What kind of effects could a mycoplasma contamination have on my cells?

"Mycoplasma contamination can: > Inhibit cell metabolism > Cause cell death > Induce morphological alterations > Alter DNA, RNA, and...

How can I detect mycoplasma contamination of my cells?

We recommend using the 20-minute MycoAlert™ Mycoplasma Detection Kit from Lonza. The simple four-step MycoAlert™ Assay is a selective biochemical test that exploits the activity of mycoplasmal enzymes. The presence of these enzymes provides a rapid...