Hello, a very similar question was asked. I provided the references in my own response. Good luck. 

https://www.researchgate.net/post/Do_M1_and_M2_macrophages_have_well_defined_surface_markers_Can_THP-1_monocytes_and_primary_monocytes_be_differentiated_into_M1_or_M2_subsets

See particularly this one. 

Food Funct. 2013 Feb;4(2):266-76. doi: 10.1039/c2fo30156c.

Characterization of polarized THP-1 macrophages and polarizing ability of LPS and food compounds.

Chanput W1, Mes JJ, Savelkoul HF, Wichers HJ.

Before polarization, you need to differentiate with PMA (5 ng/mL)

http://www.ncbi.nlm.nih.gov/pubmed/17334670

Thank you Naikei.

So your suggestion is treating THP1 with PMA (5ng/ml) for 24h, then use serum free medium make cells rest for 48h or longer (or polarize immediately?). After that, polarize the M0 cells into M1 and M2 with IFNr and IL-4(should I use medium with or without serum?). 

Could you send me the full text (Characterization of polarized THP-1 macrophages and polarizing ability of LPS and food compounds.)? Our database  can't provide download for this paper.

Thank you so much!

Dear Shujun,

I'm not sure whether the PMA-differentiated THP-1 macrophages can serve as a good model to study macrophage M1/M2 polarization. From what I understand, the PMA differentiation alone induces an M1-like state in the THP-1 cells, involving for example induction of stable IL-1beta mRNA expression (Fenton et al Journal of Immunology April 1, 1988 vol. 140 no. 7 2267-2273). 

There is an article though that made an effort to optimize the PMA differentiation protocol of THP-1 cells so that they would as closely as possible resemble primary human monocyte-derived macrophages. The optimized protocol involved 5 day resting of the THP-1 cells without PMA after the initial PMA treatment. The authors state that THP-1 macrophages cultured with this protocol "retained marked plasticity to stimulus-directed polarization", although the cells still secreted very high amounts of proinflammatory cytokines after LPS treatment compared to primary human MDMs.

Daigneault et al. Plos One 2010 The Identification of Markers of Macrophage

Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages

Hi Shujun,

Using PMA is your best option to differentiate the THP-1 into M0. There are other compounds available but they will already activate the macrophages to M1. The trick is that you should not use to much PMA. I always use 5ng/ml for 24 hrs. in the paper mentioned by Kristiina they use way too much PMA, namely 20ng/ml (I am indeed not surprised that they see M1 cell markers).

In short I would differentiate the THP-1 to M0 using 5ng/ml PMA for 24 hours. You should indeed keep the cells on -PMA for at least 48hrs after treatment. Next you should use IL-4 for M2 and a combination of LPS+IFNy for M1 (IFNY will prime them and LPS will fully differentiate them).

Good luck!

Anouk

Hi Shujun,

I forgot to mention that for M1 you could use only 10ng/ml of LPS, so that you can induce apoptosis with 100ng/ml.

For M2a I use: Arginase-1, Mannose receptor, Fizz.

For M1 I use: iNOS, TNFa, IL12, and IFNy.

I would not use IL1beta, because it is not a good M1 marker. Il1b can be both anti-inflammatory as pro-inflammatory (same goes for IL6). Also You do not have to check IL-10, because it is a M2c macrophage marker and you are inducing M2a with IL-4.

The problem regarding the high iNOS expression you observed in M2, is that you have used way too much PMA, so your M2 were previously M1 and not M0 as they should have been. SOCS-1 can go up in M1 cells depending how long your stimulus was present (can happen after 24hrs of stimulation). In my experience the optimal time-points for mRNA expression of these genes is 6-8hrs.

Again good luck with your experiment and let me know if my suggestions helped you resolve the problem ;-)

Anouk

Anouk

PMA (low dose at 5 ng/mL) differentiation takes at least 3 days. Some use 5 days. Some 3 days PMA, plus 5 days rest (medium only, without PMA). 

You may consider reducing the serum to 3%. (Change the medium with PMA, if needed on day to day basis). High serum generally has negative effects on differentiation. 

See: 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0008668

The methods papers sent from the THP-1 polarization author. 

Cheers

Thank you Nai-Kei, Anouk and Kristiina for kind help.

According to your suggestions and papers you recommended, we use PMA (10ng/ml, 5ng/ml is too low to induce our THP1 adhere to plate) treat cells (5% FBS) for 24 hours, then rest cells in complete medium (10% FBS) for 24 hours. After that, add IFNr (20ng/ml) and IL-4 (20ng/ml) polarize it for 6 hours. Fortunately, the effect of PMA seems to be decreased, but only IL-6 and SOCS1 significant higher in M1 than M0 and M2. The others markers including Arg1, IL-10, IL-1b, iNOS, TGFb and TNFa have no differences among M0, M1 and M2 groups.

The problem might be 6 hours of stimulation with IFNr and IL-4 is too short so we plan extend incubation period to 24 hours. I will share the results to you after further experiment.

Thank you all guys.

Or just try IFNy with a little bit of LPS (10ng/ml), because you really need both to get a proper M1 cell.

IFNy alone will only prime them and not fully differentiate them to M1.

Dear Zhang, please check this article:

http://www.nature.com/onc/journal/v31/n14/abs/onc2011370a.html

this simple protocol could help you.

Hi there. As you will see from the above answers, there isn't a short-cut to doing your own work and what's more, you're going to end up with a system that works in your hands, in your lab using your reagents and so on.

Decide what for you is M0, M1 and M2. Decide which things you are going to use to determine what you have - proteins? RT-PCR? miR expression? Confirm that these readouts are working and only then start poking THP-1s to see how they respond.

These cells are not straightforward to work with in a controlled manner. You'll need to pay attention to getting good cells (get them directly from ATCC, not a friend or collaborator). You'll need to keep them free of mycoplasma (so to know when they get infected by routine handling, grow them without any antibiotics. Ever). You'll need to maintain them in the same growth stage, (so make sure you count them when you seed the flask, grow them for the same number of days, and then re-seed them at the same density you started with. Don't let them over grow). Use the same plastics manufacturer. When using them for an experiment, always use cells that are less than 100% confluent and have been seeded and grown consistently with the previous experiment. If you're changing from 96 to 24 well plates, remember that it's the surface area of the well, not the volume, that's critical for your cell density calculations. If you're using LPS, make sure it's from the same organism - don't change between Ecoli and Staph, for example. If you're using cytokines, make sure they are from the same supplier (Humanzyme sell the absolutely best ones witl PBL assay services close behind - try not to use ones made in Ecoli because they may be contaminated with LPS). Make sure all your PCR reactions are within the same efficiency range (90-110%) and that you don't try and read a Ct below 35. And if you really want to be careful, be aware that many cytokines are oestradiol responsive so you probably want to use medium without phenol red.

Good luck!

G

Thank you all guys. Finally, our condition is PMA (20ng/ml, 5-10ng/ml is too low to induce complete adherence, maybe different in various brand) for 48h, then IFN-r (20ng/ml) or IL-4 (20ng/ml) for 24h. IL-6 and TNF-a are good for M (IFN-r) and MRC-1 and DC-SIGN for M (IL-4) in THP-1 derived macrophage.