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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 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...

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...

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™.

DNA-purity and Nucleofection: Can low A260:A280 ratios lead to both reduced transfection efficiency and cell viability?

Yes. To check the quality of your DNA we strongly recommend determining the A260:A280 ratio. It should be ideally 1.8 - 2.0 for a good DNA preparation, but least 1.6.

Can nicked DNA lead to reduced transfection efficiency?

Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Compare undigested plasmid to plasmid DNA digested with a suitable single cut restriction enzyme to linearize. Supercoiled plasmid will run faster than...

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...

What protocol should I use for Nucleofection of patient derived blood samples, e.g. leukemia or lymphoma cells?

We do not have a ready-to-use protocol for Chronic lymphocytic leukemia (CLL) or other blood cell derived cancer cells. The cells often have a chromosomal aberration and show the properties of a cell line. Therefore we suggest to perform an...

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.