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Do you have any information regarding differentiation of mouse ES cells after Nucleofection® with large plasmids?

It is unlikely that plasmid size would affect in vitro differentiation capability. Gene targeting constructs are often as large as 20 kb and if treated well the ES cells are perfectly capable of generating germ line chimaeras afterwards (indicating...

Does the Mouse T cell protocol work for stimulated mouse T cells?

Yes. Stimulate the cells with anti CD3 and anti CD28 antibody for 24 hours. Use the optimized protocol. Often times, this will result in higher transfection efficiency (60-70% is possible). Alternatively, you can stimulate with ConA and IL-2 for 2-3...

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...

Do you have data about the in vivo implantation of hepatocytes transfected by Nucleofection™? For how long could protein expression be detected?

There is a publication reporting on the successful reinjection of modified hepatocytes into mice.Chen et al. were able to monitor the expression over several days. This group worked with a preliminary test solution for mouse hepatocyte as the...

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.

Can I apply more than one program (transfection condition) per plate with the 96-well Shuttle® System?

With the 96-well Shuttle® system, it is possible to apply up to 96 different programs (transfection conditions) per 96-well Nucleocuvette® Plate. Therefore, optimization of Nucleofection® Conditions for a particular cell line can be...

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 protein synthesis > Induce chromosomal aberrations > Cause increased sensitivity to inducers of...

What cell number do I need per well when using the 96-well Shuttle® Device or the 4D-Nucleocuvette® Stripes?

The cell number is very much dependent on the cell type you use. For high throughput Nucleofection®, we usually start with one fifth of the optimal amount recommended for the standard Nucleofection® in the 100µl cuvette. Currently the absolute...

Are rat and mouse hepatocytes transfected by Nucleofection® still functional?

Yes, transfected mouse hepatocytes show albumin synthesis rates comparable to those of untransfected cells. Moreover the morphology and polarization of rat hepatocytes is not affected by Nucleofection®.

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...

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