HI every one,
I runned FACS on HMC3 and analyse them but I didnt get any positive marker after activation with IFN gamma and LPS. According to ATCC , HMC3 should be postive for CD14 at resting and highly postive after stimulation with IFN gamma and LPS!!. But I didnt get any postive response at all?! I have same problem as well about other markers such as HLA-DR and Cd11b!! I tested my antiobodies with postive control (monocyte) and they worked well but with HMC3 I dont get any results at FACS!! any suggestion?!
Dear Bahareh,
maybe it's worth checking out expression of the TMEM119 protein, which is supposedly specific for microglia in both mice and humans and discriminates it from blood derived macrophages that reside in the brain. Maybe it's also an explanation, why your cell line doesn't stain for macrophage markers like CD14 and CD11b.
Please check the attached product details and references:
http://www.abcam.com/tmem119-antibody-28-3-microglial-marker-ab209064.html
Article TMEM119 marks a subset of microglia in the human brain
All the best,
Michael
You may want to have a look at our paper, just published on Journal of Neuroinflammation. It is a review article on the HMC3 cell line model. It clarifies the issue about nomenclature, since the cell line has been distributed under different denominations, including HMC3, CHME3, CHME5 and C13-NJ. In addition, we provided information on the aliquot of cells distributed by ATCC, including markers in resting and IFNy treated cells.
It seems HMC3 do not express CD11b even if they have been treated with IFNgamma. Does anybody know why?
(Figure 5 from Article The human microglial HMC3 cell line: Where do we stand? A sy...
)
Did you succeed to induce inflammation in HMC3 cells??? I have tried with LPS with the concentration 10 ng/ml, 100 ng/ml, 1 ug/ml and 10 ug/ml but there was no inflammatory response. May I please know how did you that. I am using EMEM with penstep, glutamine, FCS and with or without sodium pyruvate as it blocks NF-kB pathway.
The most reliable marker of pro-inflammatory activation in this cell line is IL-6. We used a mixture of Interleukin 1 beta and interferon gamma (each at 10 ng/ml), and observed both IL-6 upregulation and increased production. You can find more details on the cell line in our article https://rdcu.be/6hXK
Thank you for the kind answer Cinzia. Can I also expect to have nitric oxide in the culture medium or it's just IL-6 up regulation??? As NO is also one of the marker of inflammation released by inducible nitric oxide synthase enzyme.
No. We could not detect any NOS2 induction, neither NO production.
Just IL-6 can be a response for something else as well. How can we be sure that it's an inflammatory response??? As COX-2 and iNOS are the main components in NF-kB pathway.
We did see upregulation of other inflammatory genes, including COX2, in response to IL1b and IFNg. The cells seem to respond differently from rodent models.
Hi Sheelu,
we used IFN gamma 10ng/ml but the reponse isnt so high but we can see the effect we want see.
Thank you for your kind answer Bahareh. Are you getting nitric oxide or iNOS response out of it???
Hi Cinzia,
Which M2 marker worked well at HMC3 surface and which cytokine? We tested CX3CR1 and CD200R at surface. CX3CR1 worked but the response between resting and stimulation with IL4 doesnt have so much diffrerence. CD200R doeant wokred at all . Can you help me about it?!
Apologies for the delay Bahareh. We checked the expression of CX3CR1 at the mRNA level only.
Hi All
I am PhD student at Purdue University. I experience similar problems of what is posted in research gate. But I would like to know the following
1) what is the purpose of ATP addition after LPS stimulation and have you detected NO using this addtion?
2) Have you checked the expression of IL-6, TNF-a, TGF-beta by ELISA. If so, what did you observe?
Please let me know as it would be useful for my research.
Thanks,
Bhavani Gopalakrishnan
Dear Bhavani,
I am facing the same problem. Unfortunately you can't get NO response and I can't say anything about the rest because I got some kind of problem with the cell line. Nothing works out and no inflammatory response.
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