Q:

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

A:
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 cell activation. Please note that the recovery medium used for these experiments did not include serum or any other supplements. There are several possibilities that could account for the activation you have observed: 1) leukocytes are very sensitive to LPS (human leukocytes much moreso than those from mouse) so it is very important for the DNA to be as pure as possible - above our normal recommendations. Namely, that there are two additional PEG precipitations to clear out residual LPS from the DNA prep on top of the endotoxin-free purification with the 1.8 OD ratio. These extra purification steps are important, since even trace amounts of LPS are sufficient to activate antigen presenting cells (APCs) like DCs, monocytes and macrophages. 2) Plasmid DNA alone can induce activation. This is a natural response of APCs to the presence of unmethylated CpG dinucleotides (i.e. characteristic of bacterial and viral DNA). So even removing all of the LPS from DNA preparations will probably not eliminate activation. A reference for this is Gene Ther. 1999 May;6(5):893-903 3) We tested only unsupplemented recovery medium (i.e. without added serum). If you have added anything to the recovery medium it will be important to verify that none of these components can induce activation. According to our R&D the cells seem fine for up to 48 hours culture without serum. However if you subsequently wish to stimulate the cells you may require serum in the medium.
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