Dear all,
I have been performing transfection of GFP-tagged plasmids into THP-1 cells using NEON electroporation system. However, the relatively low transfection efficiency suggests that I might not find an optimized electroporation protocol.
Would you guys mind provide your valuable experiences of dealing with THP-1 cells transfection using NEON system ? And whether the recovery time after electroporation is needed for further PMA induction ?
Your valuable recommendation will be highly appreciated !!
Truly Yours,
Mason
I have not used your transfection system, and I haven't worked with THP-1 cells, but I have worked with other AML cell lines (like K562 https://altogen.com/product/k562-electroporation-kit-aml-leukemia-cells-ccl243/). Usually the problem with electroporation efficiency in leukemia cell lines comes from having incorrect concentrations of the plasmid, and having the cells be at too high a confluency. If your cells are more than 70% confluent then that could be a cause of a decrease in efficiency. With regards to PMA induction, just a couple of days of electroporation should be enough to get the cells feeling fine again. If THP-1 is slow-growing, then maybe a bit more.
Hi Mason,
I have used the neon system but for BV-2 mouse microglia-like cells rather than THP-1. We did tend to see extremely poor plasmid transfection with these cells. We put this down to intact DNA sensing pathways in the cells, something which I believe is also true for THP-1. It may therefore be that it isn't that the transfection itself is the problem, but more the cells are responding to the plasmid.
Alternatives we used successfully were either lentivirus transduction to stably express a protein of interest or using the neon to transfect in vitro transcribed RNA for the tagged protein of interest. RNA transfection of either long, or short (e.g. siRNA) worked well. Best,
Ed
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