Hi everybody,
I am trying to polarize and differentiate thp1 cells to M1 and M2 macrophages. There are lots of different protocols, so I've been trying different incubation times and concentrations, but as a summary, I've done this:
To get M0 macrophages (all): 24-48h of 50 ng/ml PMA
then between 4-24h resting (medium). For M0, keep them with media.
To get M1-macrophages: 24-48h of 50ng/ml IFNg and 50 ng/ml LPS
To get M2-macrophages: 24-48h of IL-4 and IL-13 (25 ng/ml both).
In summary, when I go to flow cytometry, all of them express around 90-98% of CD11b, then I've been trying with CD38 for M1 and CD209 for M2. CD38 works quite well, not perfect, but is expressed 1 log more in M1 than M0 and M2. But, CD209, which I've read that should be expressed by M2 but not by M1, what I see is that is expressed for both populations, M2 (~70%) and even more for M1 (~90%). Does it make any sense for you? Then I've tried CD163, and any of these populations express it.
So now I would like to try other markers, but not sure which ones. Do you experience with the best markers for thp1-derived macrophages? I am afraid I am not obtaining the M2 expected population. They are really similar to M0, both morphology and markers. But I can't trust the markers yet. So not sure where is the problem.
I know the best could be to differentiate and polarize human monocytes (from the buffy coat), but I would also like to have this in vitro model.
The last question, do you have experience with another monocyte cell line? That could be polarized and differentiated to M1 or M2 populations.
I would really appreciate your help and suggestions!
For M1, you can also try CD32, CD80
CD209 is not just expressed by M2, its even expressed by M1, but to a lesser extent. Its weird that you see a strong CD209 expression in M1(90%) compared to M2 (70%).
So, For M2, as you have used IL13, try CD36, TG2 as one marker to verify its M2 phenotype.
Both the links printed above got ample information to your question on THP-1.
https://www.sciencedirect.com/science/article/abs/pii/S0022175919303461
https://www.frontiersin.org/articles/10.3389/fphar.2018.00071/full
To your last question, you can also try human leukemic monocyte lymphoma cell line U-937 or HL-6p to differentiate into M1 or M2 populations. But, THP-1 is the most common cell line utilized to study monocyte/macrophage differentiation and function. Moreover, THP-1 cells resemble primary monocytes and macrophages in morphology and differentiation property compared to other human monocytes cell lines. PMA can be used to activate U-937 cells into macrophage-like cells (M0). After 24 hours of PMA treatment, non- adherent U-937 cells became tightly adherent to the culture plates forming M0 cells. M0 cells can then be polarized into M1 phenotype by treating with LPS and IFN-γ for another 24 hours. Use of IL-4, IL-13, or IL-10 either alone or in combination for 24h will induce M0 to M2 phenotype.
I usually used CD40 and CD80, CD86 as M1 markers, and CD206 and CD163 as M2 markers.
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