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Do you recommend positive selection or depletion for the purification of Human monocytes for Nucleofection?

We only recommend the depletion method. For Example, the RosetteSep™ Isolation Kit for human monocytes [Stem Cell Technologies, Cat. No 15028]. The advantage of depletion is that the monocytes are left untouched by antibodies during the process.

Does it matter if the PBS used for monocyte enrichment contains calcium and magnesium, if the cells should be used later in Nucleofection ?

The PBS should be calcium and magnesium free to prevent clumping of cells. We routinely use PBS without calcium and magnesium for example Lonza Cat. No. 17-516F

Why do you have different Optimized Protocols for the Nucleofection of unstimulated and stimulated Human T cells?

We found that the stimulation of T cells not only changes the cells' function but also significantly alters the requirements for successful Nucleofection™. That's why Lonza developed two Optimized Protocols for transfection giving different...

How can I determine the correct centrifugation speed for my particular centrifuge and rotor?

The correct rotor speed can be calculated by measuring the maximum radius of your rotor and entering the information into the table found on the website which can be reached by clicking on the Related Link.

Is it possible to use frozen primary CD34+ hematopoietic stem cells for Nucleofection?

Yes. For cryopreserved PBMCs or enriched CD34 populations, we recommend culturing the thawed cells 1-2 hours in culture medium before Nucleofection™. Any further enrichment procedure after thawing is not recommended. Viability of frozen nucleofected...

Is the age of the mice important for my mouse T cell Nucleofection?

Yes. We recommend using mice between 6-12 weeks. Using mouse T cells isolated from younger or older animals for Nucleofection™ may result in much lower transfection efficiencies and/or viabilities.

What are the critical steps for successful Nucleofection of monocytic cell lines like THP-1, HL60 and U937?

To achieve optimal results, we strongly recommend following the Optimized Protocols in regard to cell culture conditions (medium, splitting cycle, density before Nucleofection),DNA amount and purity. Please also be sure not to exceed 90xg when...

Does the recovery medium contained in the Mouse T Cell Nucleofector Kits contain any immunogenic substances?

No. The post Nucleofection™ Recovery media contains no immunogenic factors and should not influence cell stimulation or differentiation. For Mouse T cells, some experiments have shown that after Nucleofection™ cells can be stimulated efficiently in...

I see activation of my monocytes (or macrophages or DCs) following Nucleofection. Why is this? What can I do to address this problem?

We have examined the effects of Nucleofection™ (without DNA) on these cells and have not observed significant activation. This indicates that neither the Nucleofector™ Solution, the Nucleofector™ Program nor the recovery medium are sufficient for...

I observed high mortality rates in mES cells after Nucleofection with a Cre recombinase expression vector. How can I increase the viability?

The High mortality after transfection might be due to the Cre recombinase itself, because the mammalian genome contains pseudo loxP sites (Thyagarajan B et al. (2000) Mammalian genomes contain active recombinase recognition sites. Gene....
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