Multiple protocols are available to inactivate genes in both macrophages and dendritic cells using electroporation or lipid-based transfection of siRNA pools (not to mention viral system).
However, I am having a hard time finding a protocol to transfect siRNAs in freshly isolated human monocytes; most protocols deal with the in vivo delivery of siRNAs, but I am only interested in the in vitro transfection.
I have tried it for DCs which are not that diffrerent to Monocytes and it worked quite well with efficiency of Knockdown up to 75% and about 3-5 % dead cells (siRNA from Invitrogen and Hi perfect transfection reagent from Qiagen). However, you have to optimise it by yourself (siRNA conc + Transfection Reagent) . . Happy to provide any help if needed . . . Good Luck !!!
Hi Sarah! Thanks for the link provided. Very kind indeed. Yet, have you tried yourself? Do you have any experience with it? If so, could you please comment how efficient is the transfection of siRNAs related to the cell death? Once again, thanks in advance! ;-)
Hi Nikolai,
It is a good suggestion. I appreciate it. Yet, it is my experience that freshly isolated monocytes are fragile in comparison to monocytic cell lines (Thp-1). While I have never worked with the U937 cell line, I have tried the Amaxa protocol on freshly isolated monocytes in the past and I have obtained poor results in terms of transfection efficiency and cell viability. Also, the Amaxa protocol also influences biological function of these cells such as phagocytosis, protease-dependent migration and up-regulation of co-stimulatory receptors (beyond the non-specific effects of the siRNA themselves). I thank you anyway for taking the time in considering and participating in my question forum! :-)
Geanncarlo
The easiest protocol I came across was using HiPerFect from Qiagen. I had very good transfection efficiency with a range of immune cells.
I have tried it for DCs which are not that diffrerent to Monocytes and it worked quite well with efficiency of Knockdown up to 75% and about 3-5 % dead cells (siRNA from Invitrogen and Hi perfect transfection reagent from Qiagen). However, you have to optimise it by yourself (siRNA conc + Transfection Reagent) . . Happy to provide any help if needed . . . Good Luck !!!
Yep, you’ve got a tough cell type to access. The majority of papers I have found use THP-1 cells for their siRNA experiments even when they have isolated primary monocytes for other experiments in the study. I did find a couple describing standard transfection in primary monocytes:
1. http://www.jimmunol.org/content/186/3/1638.full.pdf+html
Dhiman R, et al. c-Maf–Dependent Growth of Mycobacterium tuberculosis in a CD14hi Subpopulation of Monocyte-Derived Macrophages. J. Immunol 186: 1638-1645
Authors state that they can get 65-75% knockdown.
2. Wang X, et al. Cellular microRNA expression correlates with susceptibility of monocytes/macrophages to HIV-1 infection. Blood 113: 671-674
This paper uses miRNA mimics and inhibitors which deliver the same way as siRNA, but there is no indication of transfection efficiency.
There is one paper I found that describes the use of modified siRNA (rather than a standard transfection reagent) in primary monocytes:
Kuo P, et al. Lung Cancer-Derived Galectin-1 Mediates Dendritic Cell Anergy through Inhibitor of DNA Binding 3/IL-10 Signaling Pathway. J Immunol 186: 1521-1530
The authors show 85% knockdown of the target in the supplementary figures.
Because these papers are not focused on the method of transfection, sparse detail is given. You will likely need to do some optimization studies first (a good idea in any case since cells can behave differently from lab to lab - especially freshly isolated primary cells). These papers should get you started on reagents to investigate.
I recommend the magnetofection technology for this application, see http://www.ncbi.nlm.nih.gov/pubmed/21417829
Hi Geanncarlo,
I haven't used this particular reagent but would be interested to hear which on you chose and how you get on. I always had trouble with cell death in any transfection experiment I did and the cells I was using were quite robust. Hence why I tried to look for something specifically designed for what you are trying to do in the hope it will minimize death whilst obtaining high efficiency. Good luck!! :)
Dear Doris,
Wow! I truly appreciated your valuable input on this question forum. I will your advise to the bank! Thanks for the references and advise. Now, I will develop an strategy. ;-)
Hi Olivier,
Thanks for the novel method you recommended. I will keep in mind. Cheers!
Hi Geanncarlo,
I am also working with the same cells....Human monocytes isolated from blood and differentiate them to Macrophages. After trying several methods, we recently used the Y unit from LONZA (as the X unit is for THP-1 kind of cells) which is specifically developed for transfecting adherent cells like the ones we use (http://www.lonza.com/products-services/bio-research/transfection/nucleofector-technology/adherent-nucleofection.aspx ). The representatives from the company visited us and did the first optimization experiment. I haven't analyzed the results but they said that good results were obtained elsewhere with the same protocol.
Hope this helps.
cheers,
Aravind
Hello everyone,
I guess we got interested in this question for a bit over a year now. My students and I tried several things, and we found a method/protocol that works very well, not only in primary human monocytes, but also in human dendritic cells and macrophages. Our proposed method is out on line. Check it out (PMID: 24890643)! In any case, I appreciate everyone that participated on this forum. Cheers! ;-)
Hi Geanncarlo,
Thanks for sharing the news but i cant find the full text article....perhaps its not yet out. But it would be great if you can send us the full text or upload it on your research gate profile.
Well, it has been a while since I asked this question. Please check my profile to download the article (An efficient siRNA-mediated gene silencing in primary human monocytes, dendritic cells and macrophages) where my students and I tackled this question, and proposed a method to transfect both Macrophages, dendritic cells and freshly isolated monocytes. Cheers, everyone!
Thanks Geanncarlo, it's actually a very nice paper.
You may have thus some interest for the Viromer technology (polymer-based transfection) which unfortunately has been developed after 2014 and could not have taken part of the study.
Viromer BLUE and GREEN achieve very high KD efficiency for both PBMCs, primary monocytes, macrophages and DCs with no sign of toxicity.
You can visit this webpage for more details (scroll down for customers' data and publications).
https://viromer-transfection.com/immunology-research
At your disposal if you need more info about protocol adjustments or if you want to give it a try.
Best, Sandra
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