Hope these 2 links help

https://www.researchgate.net/post/Whats_the_best_marker_in_flow_cytometry_to_differentiate_between_M1_and_M2_macrophages

http://www.jci.org/articles/view/44490

if you want to use the cells afterwards, i.e. sort them, none of the markers mentioned above will work, as that means you need to fix/permeabilise cells. However, you can use CD163 as an M2 marker, and CD127, CD205 as M1 markers. Just check PubMed or Google, you will find enough protocols for their separation. A good starting point is the literature by David Hume.

In Supplementary Figure1 of our recent publication you will find some M1/M2 Macrophage surface markers http://bloodjournal.hematologylibrary.org/content/early/2013/10/29/blood-2013-05-501494/suppl/DC1. Hope that helps!

Would this be different when looking at rat macrophage phenotype?

Some of the markers mentioned above are also found on other myeloid cells (dendritic cells, neutrophils, basophils, suppressor cells…), so it depends on the source of your macs. If it is from tissue, I doubt you can have a complete purified fraction using only 1 single marker. You may need to add a cocktail and discard non-macrophage populations.

YM1,2, INOS and arginase are not appropriate markers for human macrophages. They work for mouse. If you are producing M1/M2 in culture then I would suggest CD80 staining for M1 and CD163 (high) for M2.

CD163 +/high can be used somehow as an M2/preM2 Mac.

CD206 (MMR) is a pro-M1 marker (See Fig1. http://bloodjournal.hematologylibrary.org/content/107/12/4930.long) and others, including our own lab experience.

Again, CD80 will be expressed on dendritic cells and also macrophages activated with TSLP (which are pro M2). As said before, you may require a combination of markers. Try to use CD64, CD68 and/or CD38 which are poorly or not expressed in the majority of resting myeloid cells other than macrophages and then check CD163 and CD206. If you have activated the cells with IFN-gamma or IL-4, you may not use the aforementioned macrophage markers (or maybe only if you use the whole lot)

The references previously given (The Blood Oct 2013) uses M-CSF derived macrophages from human monocytes which, in my understanding, differentiates those monocytes into pro M2 macrophages instead of M0.

(http://www.pnas.org/content/101/13/4560.long)

Some markers are tissue specific for macrophages so it might different. Typically, M1 macrophages will be iNOS+ (some tissue CD11c+ but be careful because it is a dendritic cell marker). M2 macrophages activated by IL-4 and/or IL-13 will be CD206+ and CD163+. As a general marker to detect all macrophages you can use CD68. Good luck!

Cell surface expression of CD40 is increased in M1 macrophages (LPS/IFN) compared to M2 (IL-4/IL-13) and CD36, CD206 and CD163 are increased in M2 compared to M1.

Intracellular MIP1a for M1 marker. DC SIGN (togheter with a marker for macrophages, such as CD68) for M2a macrophages.

Hi I have read in a paper, I think this paper will really help you a lot.

The Journal of Immunology, 2009, 182: 6237–6246.

Titled: M1 and M2a Polarization of Human Monocyte-Derived Macrophages Inhibits HIV-1 Replication by Distinct Mechanisms1

Edana Cassol,2*†‡ Luca Cassetta,*† Chiara Rizzi,* Massimo Alfano,* and Guido Poli*†

The capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines and other extracellular stimuli. In this study, we demonstrate that cytokine-induced polarization of human monocyte-derived macrophage (MDM) into

either classical (M1) or alternatively activated (M2a) MDM is associated with a reduced capacity to support productive CCR5-dependent (R5) HIV-1 infection. M1 polarization was associated with a significant down-regulation of CD4 receptors, increased

secretion of CCR5-binding chemokines (CCL3, CCL4, and CCL5), and a >90% decrease in HIV-1 DNA levels 48-h postinfection, suggesting that the inhibition occurred at an early preintegration step in the viral life cycle. In contrast, M2a polarization had no effect on either HIV-1 DNA or protein expression levels, indicating that inhibition occurred at a late/postintegration level in the viral life cycle. M2a inhibition was sustained for up to 72-h postinfection, whereas M1-effects were more short-lived. Most phenotypic and functional changes were fully reversible 7 days after removal of the polarizing stimulus, and a reciprocal downregulation of M1-related chemokines and cytokines was observed in M2a MDM and vice versa. Since reversion to a nonpolarized MDM state was associated with a renewed capacity to support HIV replication to control levels, M1/M2a polarization may represent a mechanism that allows macrophages to cycle between latent and productive HIV-1 infection.

this is the paper I was refering to about M1 phenotype (intracellular staining for CCL3.

We recently pusblished another paper showing DCSIGN as a specific M2 marker.

Hi Abdullah,

I have been working with human macrophages, obtained from monocyte differentiation during 7 days. I use M-CSF and GM-CSF in order to obtain different anti- and proinflammatory macrophage subsets, respectively. Based on my experience, you can select human M2 macrophages by CD163, which should be negative in M1 macrophages population. Also CD14 mean will be usefull to discriminate between both macrophages subsets, so M2 are really bright, and M1 should be totally/almost negative. In the case of M1 macrophages selection, perhaps you can choose costimulatory molecules as CD40 or CD80, MHCII molecules HLA-DR or HLA-DQ, but I agree with iNOS could be the best choice.

The question is what kind of sample are you starting to proceed with the isolation, and the activation/steady state of the cells, so depending on these factors some markers could be the "gold standard", or simply these markers would be expressed and shared by different cell types (in the case of DC, M1 and M2 there are several molecules expressed in both three cell types).

As a comment, in my experiments DC-SIGN (CD209) is almost undetectable on surface of M2 macrophages.....there are several and recents reports suggesting this PRR as an alternative marker for M2 macrophages, but in my hands only in a few number of donors analyzed had M2 CD209+, and MFI was really low......can anybody with experience with this molecule give any suggestion with this trouble?

Hope it helps, and thanks in advanced!!