Hi Xiaojie Yan, it has been asked x thousands times on that forum already.

Shortly, first it's not the reagent the problem. And it's not the plasmid delivery the problem, but its processing once into the cells. Immune cells react to foreign introduced DNA by simply destroying it. No chance to reach the nucleus, to be transcripted, and then finally be translated.

Maybe a small chance with models like RAW but not with primary cells.

An alternative is to transect mRNA... Much more faster and powerful but of course only transient.

And not so easy as plasmid handling, you would to produce mRNA by IVT or buy it to a supplier.

And then you would need an adapted transfection reagent such as our Viromer mRNA.

Hope that helps a bit, but my pleasure to give more details if needed.

Best, Sandra

[email protected]

Sandra Lagauzère ,Thanks for your reply. I agree with your answer greatly. However, for the article I mentioned above published in ACS Nano and Nature Communications, the authors said that they had succeed in transfection of BMDMs with plasmids using Lipofectamine reagent with a great positive result, of course including mRNA, but I failed. I wonder how they can make it success?

Hey Xiaojie.

i completely agree with Sandra. Immune cells like macrophages are "built" to detect foreign DNA. They react with destruction of the DNA, a pro-inflammatory response or even with cell death. RAWs are a macrophage-like cell line, far away from what a ex vivo or in vivo macrophage can do, but they are easier to transfect.

I am always very sceptical, when researchers "accomplish" to transfect macrophages with DNA sucessfully without tremdendous activating side effects (because that is how macrophages react to foreign DNA in vivo).

We were confronted with the same problem as you a while ago, so we recently established a protocol for transfection of immunologically silenced mRNA in bone marrow-dervied and pertitoneal macrophages. If you like, please have a look at:

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA, November 2019, Journal of Visualized Experiments.

For further information about the do's, don'ts and difficulties of transfection macrophages, please also have a look at:

Zhang, X., Edwards, J. P., Mosser, D. M. The expression of exogenous genes in macrophages: obstacles and opportunities. Methods in Molecular Biolology. 531 123-143, doi:10.1007/978-1-59745-396-7_9, (2009).

Guo, X. et al. Transfection reagent Lipofectamine triggers type I interferon signaling activation in macrophages. Immunology and cell biology. 97 (1), 92-96, doi:10.1111/imcb.12194, (2019).

Van De Parre, T. J., Martinet, W., Schrijvers, D. M., Herman, A. G., De Meyer, G. R. mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages. Biochemical and biophysical research communications. 327 (1), 356-360, doi:10.1016/j.bbrc.2004.12.027, (2005).

Kaestner, L., Scholz, A., Lipp, P. Conceptual and technical aspects of transfection and gene delivery. Bioorganic & medicinal chemistry letters. 25 (6), 1171-1176, doi:10.1016/j.bmcl.2015.01.018, (2015).

All the best,

Marc

Marc Herb ,Thanks indeed! You convinced me, and I will have a try to transfect the BMDMs with mRNA. Thanks again! Thank you very much!

You are very welcome

Marc Herb , hi, Marc, I have tried to transfect BMDMs with EGFP reporter mRNA and lipofectamine 3000. The result was unsatisfied and little cells turned to be green, besides I found the shape of cells turned to be round and some cells were dead after transfection. I noticed that the transfection reagent you used was jetMESSENGER, I don't know whether the reagent played an important role in this result. Could you please help me to find the problem?

My detailed protocol is shown below:

200,000 BMDMs cells per well were seeded in 24 wells plate overnight. Before transfection, the BMDMs medium were replaced with 500 uL DMEM without serum. 1 uL lipofectamine was added into 24 ul DMEM, 500ng mRNA and 0.5 uL P3000 were added into 24 ul DMEM. After incubation for 5 mins, both of them were mixed and incubated for 15 mins. And the mixture were drop-wise added into the well. After 5 hours incubation, the medium were replaced with BMDMs medium (DMEM with 10% serum and 25 ng/mL M-CSF).

Before the replacement of medium, I found that the cell began to be round, and some cells floated.

I am looking forward to your early reply!

Hey Xiaojie,

your protocol sounds perfectly fine to me concerning cell handling, medium, incubation and amount of mRNA.

To answer your questions, yes!, the jetMESSENGER was the only mRNA transfection substance that sucessfully delivered mRNA into primary macrophages.

Lipofectamine and other delivery vehicles did either not work or the cells died at least with primary macrophages (I consider immortalized macrophage cell lines like THP1 or RAW not as primary macrophages).

During generation of mRNA you should also consider removing any traces of DNA templates (because this also induces macrophage death).

Additionally, you can use 5-methyl-CTP and pseudo-UTP, which are bases that reduce immunogenicity and enhance stability of the mRNA.

Moreover, you can dephosphorylate the 5’-ends of the in vitro transcribed mRNA by Antarctic phosphatase to prevent recognition by the RIG-I complex.

All of these steps together led finally to sucessful macrophage transfection, but it took long to get it right.

I hope that further helps you.

Please keep me informed and all the best,

Marc