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Do you recommend a purification method to purify specific blood/immune cell populations before Nucleofection®?

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

I want to stimulate human primary T-cells with anti-CD3 and anti-CD28. Do you have a detailed protocol for this?

Yes, the detailed protocol is part of our 4D-Nucleofector® Protocol for stimulated Human T Cells.For optimal stimulation of the transfected cells we recommend coating 96-wells plates for stimulation [Nunc Immuno™ Plate C96 Maxi Sorp™, Cat. No. 430...

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 in Nucleofection® experiments?

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...

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.

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

Unfortunately, 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...

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...

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...

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...

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™.
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