I am currently attempting to optimize ViromerRED transfection in THP-1, H9, and MCF7 cells. Our initial transfections with the first batch was promising, but the new viromer we received doesn't seem to be working at all. I have contacted the company to see if there might be an issue with the new lot. Our first transfection attempt showed a transfection rate of about 6% GFP, however now we are seeing absolutely no GFP. In the mean time, has anyone been using viromer successfully and if so, any tips or recommendations?
Thank you for your time.
Hey Anastasia,
we tried ViromerRED (and also a few other transfection reagents) for mRNA transfection of peritoneal and bone marrow macrophages as well as MEFs and it did not work. We could not detect any GFP expression (about 2-4%). Instead cells started dying with increasing transfection reagent concentration.
But we recently established sucessfully a protocol to transfect mRNA with the JETmessenger transfection reagent. It worked for GFP and for other mRNAs (IKK, NEMO) in all tested cell types. For a detailed protocol please have a look at:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (Pending Publication), e60143, In-press (2019).
Please feel free to ask me any further questions.
I hope I could help you,
All the best,
Marc
Hello, I am Sandra from the Viromer Tech support!
First, I would like to directly answer the title question > YES many people have optimized Viromer RED (and others) in specific cell lines.
Because all cells are not responding the same to the standard protocol, it's crucial to perform some optimization as suggested in our manual (same thing for other reagents).
And not only the cell type... each combination of plasmid or RNA you want to deliver, experimental conditions, cell density etc may require adjustments to have the best of your transfection.
also good to make a difference between the efficiency of the delivery (are you sure the DNA or RNA does not go into the cells?) and the desired cellular action (what happens to the delivered material, does it reach the nucleus, etc). That why controls are important.
OK, we are not as big as Life Tech or Polyplus, but the Viromers have shown great results and proven efficiency through >220 publications citing use of them in only 5 years of presence on the market. Please, check attached table featuring these publications. First pages are dedicated to immune cells, and going through you will find precious tips to adjust protocols.
As Marc Herb reports, mRNA is also the only alternative with immune cells. We generally advise against plasmid transfection as well, and the new Viromer mRNA is an excellent reagent for that purpose (clearly outperforming JetMessenger in our hands!). I am very sorry to read you were unsuccessful with your macrophages... many other immunologists are very happy with Viromer RED and the new Viromer mRNA.
Among others, I am sure you will have interest for this very nice publication citing use of Viromer RED:CCL3 mRNA for recruitment of monocytes from blood to the peritoneal cavity, and then for reprogramming them into M1 and M2 macrophages with co-expression of IFN-gamma and IL-4, respectively.
Article Exploitation of Synthetic mRNA To Drive Immune Effector Cell...
Anastasia Ritchie > I found that you actually contacted my colleague Bettina about the lot issue (yesterday!!). Would have been great to go ahead with her recommendations before asking on the RG forum... but I understand you. ;-)
We can naturally replace you new Viromer RED if there is any suspiscion with the received material.
For MCF7 and THP-1, there is probably something to do, including switching to mRNA if you work with plasmid transfection as Marc Herb suggests. Or simply to really test different conditions, and maybe use a very easy-to-transfect cell type as control, like HEK or HeLa, to be sure it's cell-dependant.
Last thing, as my colleague told you, it's more tricky for T-cells. There is no chemical transfectants working for these cells (Viromers but same for the others). We assume it's something related to the recycling of materials once into the endosomal compartment. Electroporation and viral transduction are the only methods working.
Hope that my answer clarifies a little bit things. At your disposal if you have any other questions.
Best,
Sandra
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