Telling apart M1 and M2 macrophages by surface phenotyping is very difficult. I've been trying to do it myself. Macrophages are defined by their functionality rather than surface flow cytometry or colorimetric properties. Though a new paper out this week by Gundra et al have had a look at several common surface markers in M2 cells (doi: 10.1182/blood-2013-08-520619) such as CD206, PD-L2, FIZZ1/RELMalpha.

The paper also includes several PCR targets and gene clusterine analysis of M2 macrophages that may help you find a way to accurately confirm that their activation is complete.

Commonly accepted broad markers however for M1 MOs are CD68+ CD80+ and M2 CD68+ CD163+ in addition for F480 and CD11b in both, other markers are down to if you want to look at tissue resident M2 or monocyte inflammatory M2 cells which the paper also goes into.

Personally for IDing M1 and M2 macrophages I go on their functionality which is really a better measure so I do intracellular flow cytometry for pan markers F480/CD11b and then several products like CD68, CD80, CD163, iNOS, IL10, IL12, Arg-1, Fizz, INFg and CCR7.

Hi Poornima,

I agree with Samantha, measurement of surface markers can be a bit complicated in several aspects, including availability of flow cytometers and appropriate antibodies. If all you can count on is a colorimetric method then I would suggest TNFalpha and IL10 production (ELISA), for instance. By definition, M1 macrophages will produce TNFa when stimulated with LPS and M2 macrophages won't. Also, M2 macrophages will produce IL10 even in absence of stimulations whereas M1 won't.

Good Luck.

Hi Poornima,

You can measure nitric oxide for M1 using Griess reagent. Similarly, for M2, to confirm arginase activity you can measure urea in cell pellets. There should be commercially available kits for both.

Thanks to all for their suggestions

Please see this paper where membrane-associated proteins have been identified that can discriminate M1 and M2 macrophages: Article Unique Proteomic Signatures Distinguish Macrophages and Dend...

Please also see our recent study that utilized this information in order to identify macrophage polarity: Article Nanomedicine-driven neuropathic pain relief in a rat model i...

I hope this helps.