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My Nucleofector Solution was frozen. Is this a problem?

As long as the supplement was not added to the Nucleofector™ Solution, then there is no risk of any damage to the solutions. Even long-term storage of several months did not alter the performance of the Nucleofector™ Solution. However, if 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™.

I have a GFP construct that I use and usually get anywhere from 50-80% GFP positive cells using FACS analysis while I get only 30-40% using a GFP-NFAT fusion protein driven by the same promoter. Do you have any suggestions?

The Amaxa™ Nucleofection™ Conditions are optimised for each cell type, the solutions and programs are cell type dependent and not vector dependant. The expression of the fusion protein depends on the localization of the protein transcription,...

I currently work with some adherent cell lines, such as 293, which are grown in DMEM media. I have noticed that the mortality seems to be high after Nucleofection. Do you have any recommendations?

For some adherent cell lines we strongly recommend using RPMI instead of DMEM medium. As described in culture conditions of the cell line optimization kit protocol as well as the NIH3T3 (DSMZ) protocol, using RPMI instead of DMEM as "transfer and...

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).

Can I use the Nucleofector Technology for RNAi applications? How do I start?

Yes. The Nucleofector™ Technology can be easily applied for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type specific Optimized Protocol for DNA vectors (cDNA, shRNA or miRNA expressing plasmids)...

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.

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...

Can I order any of the Nucleofector Kit components separately?

No. The Nucleofector™ Kits are only available as full kits.

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).
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