Hello

There are several cell surface markers, I will mention to you some of which :

for monocytes :

low expression ( CD9- CD35-CD49a-CD49b-CD49d )

High expression ( CD10-CD11a-CD11b-CD11c-CD13-CD14-CD45RB-CD45RO)

for macrophages :

low expression ( CD9- CD49a-CD49b-CD49c-CD49d )

High expression ( CD10-CD11a-CD11b-CD11c-CD13-CD14-CD45RB-CD45RO-CD49e)

please , read the references in the attachment to find out more from the cell surface markers

Best wishes

In case of M-CSF differentiation, cell surface staining of integrin alphaV worked well for us:

http://www.ncbi.nlm.nih.gov/pubmed/16473416

http://www.ncbi.nlm.nih.gov/pubmed/18840709

Our lab used CD11b a lot. It seemed good enough.

F480 is expressed only on macrophages and not on monocytes

Well F4/80 is a murine marker, do you mean the human equivalent EMR1 Uma?

CD11b in combination with scatter works well.

My anderstanding is that  the human EMR1 is less cell type specific than the murine F4/80. 

For human macrophages the classics CD11b, CD64 (and CD68) works pretty well. 

Be Careful on which macrophage type and model you are working with. Blood monocyte derived Macrophages, THP1 or other cell lines... they don't all exactly maturate the same way and thus don't all express the same markers...

Thanks for the helpful comment, much appreciated!

Using CD11b, CD64 and CD68 as markers to differentiate, I understand these are upregulated? I have read that the two monocyte subsets, i.e. CD14+CD16+ and CD14+CD16-, differ in CD64 expression, i.e. present and absent, respectively.

I would be using human blood monocyte-derived macrophages by the way.

I have to admit that I wasn't aware of CD64 expression in monocyte subsets. Then I think that your best bet is to start with CD11b and carefully check on size and complexity subsets on the flow cytometer as some subset of NK and neutrophils can express it. But if you work with well sorted (magnetic beads, or FACS)  monocytes you should not see any of these two other cell types in your cell preparations.

• There is an article by Zawada Ma et al, published in Immunobiology 2012 that will give you more information on monocyte immunophenotyping besides CD14/CD16. 
• Macrophages have distinct subtypes also. Their phenotype may change in some tissues?? 

Indeed, I work with MACS-isolated monocytes. Thanks for the tips!

Indeed physical parameters help to distinguish between monocytes and macrophages and besides CD14 (which gets downregulated during macrophage differentiation) and CD16 (which gets upregulated), I should definitely use CD68 and CD64 as bona fide markers for macrophages, especially if you're working on purified isolated monocytes.

If you'll also activate your macrophages with specific stimuli, then you'll have to consider M1 or M2 markers, also according to the stimulus used.

Human monocytes express high levels of CD14 and low CD16, while macrophages express low CD14 and high CD16. Additionally, CD68 is a very classic marker for human macrophages. However, it is intracellular and you must permeabilize the cells if you'd like to do flow cytometric analysis. If interested, you can also use CCR5 as monocytes have very low expression of the chemokine receptor, while macrophages express very high levels. I also know some people use CD71 to distinguish between monocytes and macrophages - as it is abundant in macrophages but minimally expressed in monocytes. Good luck, hope this helps!

Thanks for the additional marker suggestions Dionna, I will have a look into it.

CD68 is the best marker of macrophages and CD14 is used for monocytes. However, CD44, a marker for phagocytes can be used for macrophages in addition to CD68. Monocytes generally not express CD44, because they are not phagocytic in nature.

Thanks,

Debanjan

if you see the classical practises, then CD68 is the best markers, However, recently there are some reports where, some leucotrines have been tried and found satisfactory for such studies.

I would suggest EMR1 (the mouse F4/80 human homolog) which is higly expressed in mature macrophages and only at low levels in circulating monocytes. Also this review can be useful: http://www.sciencedirect.com/science/article/pii/0022175994900051

Hi Alasdair,

I'm also interested in macrophage markers. This 25F9 seems really promising. Do you know whether the protein it recognizes is also expressed on MDDCs? Because I'd need to differentiate between monocytes, macrophages (MDMs) and MDDCs.

Thanks

Hi Jorrit,

What type of experiment were you referring to? Are you differentiating them in vitro or do you want to distinguish both cells in tissues? I do not completely agree with Dionna regarding monocytes, because you also have a CD14lo monocyte subset. Human monocyte subsets are: CD16+CD14-, CD16+CD14+, CD14+CD16-. The macrophage markers she mentioned are indeed very helpful in distinguishing the two. 

I agree with Anouk, and related to her answer I have a little question: Which of these monocyte subsets are related to M1 or M2 machrophage phenotype? That is to say, M2 machrophages are CD14+ or CD14low and CD16+ or CD16-?

They are CD14lo and CD16+ Juan ;-)

Thanks! :D you can provide me an article that supports this? 

Hi Anouk and Juan,

Thank you for your contribution. Anouk, it considers in vitro differentiation of monocytes.

Thanks Jorrit. I will get back to you on that. We are trying the opposite right now. We are trying to keep the human monocyte subsets in culture (alive and without differentiation to a macrophage). i will let you know very soon which FACS panel we use to check our cells ;-)

Thanks Anouk, that's very kind!

do you have any updates about the best markers to differentiate by flow between monocytes and MDM differentiated in vitro using 25ng/ml MCSF in RPMI-glutamax complete medium?

N.B: I work on human PBMCs. thank you!

Anyone have any updates on this? We are interested in separating monocytes from blood-derived macrophages in placental blood / cord blood. Thank you.

Following

Dear all,

it totally depends on the diffraction protocol you are using (if you are using M-CSF you'll get CD14+ Mo-Mph but if you are using GM-CSF you'll get CD14- Mph).

Abdullah Al Tarique

Thank you in advance for your answer.

"CD68 and CD71 are unique in human macrophages" is a misleading information.

CD68 can be expressed by macrophage/monocytes (including Kupffer cells and microglia), basophils, dendritic cells, fibroblasts (Verh Dtsch Ges Pathol 2003;87:215, Ann Rheum Dis 2004;63:774), Langerhans cells, mast cells, myeloid cells, CD34+ progenitor cells, neutrophils, osteoclasts, activated platelets and B and T cells (this information is taken from https://www.pathologyoutlines.com/topic/cdmarkerscd68.html and on daily basis is our observation in flow lab). It is a lysosomal protein and can be expressed even by epithelial cells with some stimulation.

CD71 is an activation marker, a transferrin receptor expressed by many cells. Number of this receptor increases on cell surface of actively proliferating cells, and it is essential for iron transport into proliferating cells (benign and malignant). CD71 is highly expressed in placental syncytiotrophoblast, myocytes, hepatocytes, basal keratinocytes, endocrine cells of the pancreas, spermatocytes and erythroid precursors (https://www.pathologyoutlines.com/topic/cdmarkerscd71.html). We use this marker for diagnosis of AML M6 in the hematology laboratory.