You can use 10-100 ng/mL PMA for 2-24 hours with washing and subsequent resting in fresh medium for 24-72 hours to differentiate THP-1 cells to macrophages. This is only helpful if you need a classically activated M1 macrophage phenotype cell. If your assays are sufficient with a monocyte type cell line then you do not need to differentiate them at all.

50 ng/ml PMA for 24 h

When I started culturing THP-1 cells in our lab I had done studies in primary human monocyte-derived macrophages and I wanted to find a cheaper & easier-to-handle macrophage model for a part of my project. Whether you need to perform the PMA-induced differentiation of the THP-1 monocytic cells to a macrophage-like phenotype depends entirely on the hypothesis you would like to test. As Yaron mentioned, after PMA differentiation the THP-1 cells are polarized towards proinflammatory activation. For example, the cells stably express high levels of pro-interleukin-1beta without the need for additional stimulation with bacterial components, which was relevant to my own studies.

The protocols used for PMA-differentiation of THP-1 cells vary immensely. I wen´t through a large body of literature when I started working with these cells and found that perhaps the most common protocol involved 2-3 day culture with 100 nM PMA, after which cells were used either immediately or after 1-2 days rest in PMA-free medium. If you want to mimic primary human macrophages as closely as possible, the following open access paper thoroughly compares different THP-1 differentiation protocols to primary cells characteristics:

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

Daugneault 2010 The Identification of Markers of Macrophage Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages

Macrophages represent the pro-inflammatory state of body. I used 100ng/mL PMA in RPMI 1640 + 10%FBS to differentiate THP-1 cells and the macrophages are mostly ready after 48 hours, however, most people say it would be 72 h. But 48 h is good enough. Then wash them with hank's buffer and incubate in serum free media overnight (resting phase). After this step, you can start your treatment study.

Kristiina, Yes, PMA stimulated THP-1 (TDM - THP-derived macrophages) do express proIL-1beta right after stimulation. However, after 5-7 days of rest proIL-1 beta is dissapeared ( and some other monocyte signatures) and now is possible to stimulate these cells with other ligands to evaluate cell activation. This is our model to stady inflammasome and proIL-1 beta is one of the major marker I am looking to decide when to use PMA in my experiment.

When you want to differentiate THP-1 cells in "M0" Macrophages with high viability you should use 10 ng/ml for 48 h and than a resting time for 48 -72h before stimulation.

Have a look on these papers:

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

http://www.ncbi.nlm.nih.gov/pubmed/24269601

I agree with the above responses but bear in mind that THP-1 is a cell line and it is always better to use primary monocytes or primary macrophages if you can.  You will get much more physiological gene expression profiles in the primary cells.  Also remember that the alternative name for PMA is TPA (tumour promoting agent).  You should use this chemical carefully if you plan to do experiments with it.

Very valid point of Jonathan. PMA widely and no specifically activates cells. We stimulate by PMA only THP  stably expressing or knocking down some genes and in all other cases use primary monocytes, macrophages and MDM. This is more physiological. 

-I used 100ng/mL PMA in RPMI 1640 + 10% FBS to differentiate THP-1 monocyte cells and the macrophages are mostly ready after 48 hours

I have worked with THP1 for a time, and there are many factors that affect it is own phenotype and its response to PMA. One factor is the culture condition (confluence) of THP1, which has a critical role on THP1 phenotype and it's response to PMA as demonstrated by (Aldo et al. 2013).

but by comparing different articles I believe that THP1 cells confluence is not the only factor that affect phenotype of THP1.

so what I mean, for differentiation, I believe there will be no fixed protocol, but the conditions should be optimised to get to optimum cell density, optimum PMA concentration, incubation time and resting period.

THP-1 monocytes are differentiated into macrophages by 24 h incubation with 150 nM phorbol 12-myristate 13-acetate followed by 24 h incubation in RPMI medium.

thanks so much for all your response.

The washing step after differentiation with pma, how gentle do we need to be.

Can we wash like it is RAW264 macrophages? Or we need to be very gentle?

Karthik Subramanian do you use RPMI + 10%FBS or you supplement this with L-glutamine?

Karthik Subramanian you can supplement with L-glutamine as well to boost the proliferation. For differentiation I added 150nM PMA for 24h.

Dear expertise,

Could anyone help me, how to prepare adherence cell line from THP-1 suspension cell line ?

Hello, It was tricky to set up the protocol to differenitate THP1 and U937 given that you find different conditions in the literature. NOw we use 100 to 300 ng/mL for 24 hours. Then change the medium and wait 24 hours before doing the experiment. You need to be carefull with PMA, as it is stable in stock solution for about 6 month at -20°C. We used individual aliquotes of stock solution to make the working one and never reuse them. Also pay attention, there might be variations between PMA batches. It is worth testing the new one against an old one and make a titration to check the concentration.

Good luck

PMA has time and dose-dependent effects on monocytes. it is better to use lower dosages with longer (48 h) incubation with PMA. PMA is toxic to monocytes

Article Investigation of Cytokine and Midkine Responses of Human THP...

Calculate the volume of PMA based on your total volume with number of cells in the media and incubate for 48hrs.