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What is the ratio of Supplement to Nucleofector solution?

The ratio is 1:4.5 (500µL's of Supplement is added to 2.25mL's of Nucleofector solution). For a single reaction, use 18 ul of supplement plus 82 ul of solution to make the 100 ul.

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...

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™.

What are useful methods to enrich/purify specific hematopoetic cell populations before Nucleofection?

Specific blood cell populations can be enriched/purified for Nucleofection™ by MACS™ selection Kits (Miltenyi Biotec GmbH), RosetteSep™ Kits (StemCell Technologies Inc.) or Dynal™ beads (Invitrogen Corporation).

Is there an advantage of the MACS Selection Kit (Miltenyi Biotec GmbH) compared to the RosetteSep Kit (StemCell Technologies Inc)?

The RosetteSep Kit is less expensive than the MACS Selection Kit. The MACS Selection Kit results in a higher purification (up to 97-98%) of the specific cell population.

Do you recommend a specific purification method for the selected hematopoetic cell population before Nucleofection?

For obtaining highly purified cell populations, we always recommend using the MACS™ Selection Kit (Miltenyi Biotec GMbH).

MACS Selection Kits (Miltenyi Biotec GmbH) offer the opportunity to purify specific blood/immune cell populations by positive selection via antibody-binding or by negative selection using depletion. Which MACS purification method is preferred by Lonza?

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

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.