Dear all-
What would be the best way to transfect macrophages/dendritic cells in vitro with plasmids for reporter assay or flourescent imaging with a good transfection efficiency without causing too much toxcitity that can be used for immortal/primary cell lines?
I agree with Won Lee's summary. Macrophages and DCs tend to be terminally differentiated and don't undergo much proliferation, making it difficult for them to incorporate transfected DNA into their genome. If possible for your studies, your best bet would be to transfect monocytes that can subsequently be differentiated into DCs or macrophage by culturing with the appropriate cytokines.
Hi Woojong
The only way I was able to transfect macrophages was using lentiviral transduction on BMDM during the differentiation stages. However the exogenous DNA can activate some pathways which can affect your end result. Another possibility is to transduce Asc KO macrophages because you won't get cell activation due to DNA insertion.
For the RAW cel line you can simply transfect them with Lipofectamine LTX. As for mouse bone marrow derived macrophages you van use either Lenti or Adeno. For me Adenovirus gave the best results though.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774191/
Hi Anouk
I never used adeno, could you answer me one thing please? Did it activate your macrophages?
Hi Andre,
Yes it activated the BMDM a bit, just like the lentivirus. Some of the inflammatory markers were increased. Mainly INFy. So it very much depends on what you are studying. I was studying SDF1 after Nur77 overexpression at the time which was not affected bij the control adenovirus.
At the moment I am trying to transduce them with retrovirus to see if that gives less activation. I am also trying to Transduce bone marrow and thereafter differentiate them to macrophages to see if that is a better way to go.
Hi Woojong and followers,
Forget plasmid transfection! Macrophages and DCs have actually a too strong DNase activity in the cytosol (e.g. AIM2/IL or cGAS/STING/IF enzymatic cascades).
Why not transfecting directly mRNA?
It works pretty good with Viromer RED, a polymer-based reagent featuring an active endosome escape like a virus does.
Please, let me know if you wanna try.
Bests, Sandra
Hi Sandra,
I was wondering how long does the over-expression last? And if have you also tried this method for a BMT?
In my case I am doing knockdowns so unfortunately cannot use this technique. Unless it works with shRNA as well?
Still it looks pretty cool !!! Thanks for sharing ;-)
All the Best, Anouk
Hello Anouk,
Thanks for your interest!
Usually, the transient expression from mRNA in cell culture peaks around 18-24h and decreases over time afterwards. But it depends also on the cell type.
So far, we have no feedback for bone-marrow mesenchymal stem cells with Viromer RED, sorry. But in theory, it could work as well. Also with shRNA. If you are motivated, your tests would be very welcome!
Concerning silencing via siRNA transfection, it works good for most of mononuclear phagocyte cell lines with Viromer BLUE or Viromer GREEN (>80%, see attached file for macrophages, monocytes, PBMCs, or the publication for cGAS silencing in DCs).
Bests,
Sandra
http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1005546
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