Macrophage polarization problem? CD206 didn't go up with IL-4 while went up significantly with LPS?

Recently, I analyzed the expression level of CD206 in THP-1 derived macrophages using flow cytometry.

But, the problem is cell sorting. I don`t understand why the dot plot made a continuous shape.

because of that problem, I can`t be gating of live cells for analysis. and I can`t get any change in CD206 expression levels.

Please, tell me your advice.

Ps, The antibody work very well in immunofluorescence analysis

PMA (100nM)-treated THP-1 cells cultured with IL-4/IL-13 (each 20 ng/ml) for 48h

Buffer for washing and analysis: 10% FBS, 1% NaN3 in PBS

Antibody binding buffer: PBS (1 hour in RT)

Antibody: CD206 (MMR) Monoclonal Antibody (19.2), PE (Invitrogen)

I was wondering how I could induce the M2 phenotype and specifically CD206 expression in RAW264.7 cells? I read that treating RAW264.7 cells with IL-4 did not lead to expression of CD206. If IL-4 does not lead to expression of CD206, then I was wondering if there is another way I can induce expression of CD206 in the RAW264.7 cell line. Thank you in advance!

Hi everyone,

I am working with THP-1 for my PhD project and I am having problems after the treatment with PMA.

I follow this protocol:

1. Seed the cells in 6-well, density 3x10^5 cells/ml

2. After 24h from seeding, add 10 ng/ml of PMA (24h treatment)

3. After 24h from the PMA treatment, change the media and leave the cells to rest for 72h

4. Following this resting period, to obtain M1 I treat the cells with 100 ng/ml of LPS and 20 ng/ml IFN-gamma, instead for M2 with 20 ng/ml of IL-4 and 20 ng/ml of IL-13

The problem is that, at the end of the resting period (72h), most of the cells are in suspension and the ones adherent have an abnormal phenotype, like they are dying.

As culture media, I use RPMI 1640 + 10% FBS no heat inactivated + 1% Pen/Strep + 1% L-Glut. Would anyone have any advice for me to solve this problem?

I am differentiating macrophages from THP-1 cells using PMA. Following PMA differentiation, I will be aiming to obtain M1 and M2a macrophages (by adding LPS, IL-4 etc).

Having read many publications regarding macrophages, I'm getting slightly confused. I am trying to select markers for flow cytometric panel to differentiate between different populations of macrophages e.g.

CD11b is a pan-macrophage marker which can be used to make a distinction between macrophages in general from THP-1 cells. Then I thought of using for instance CD86 and CD80 to select M1 macrophages. Similarly, I'd select CD163 and CD206 for M2a.

I'm not sure whether this is the best strategy to do so. I've seen multiple gating strategies and ways to make distinction between different populations of macrophages. I also understand they can be divided functionally or phenotypically. Any suggestions or good publications/protocols in this area would be much appreciated.

Hello,

I have been trying to polarize PBMC derived monocytes into M1 macrophages, using varying concentration of LPS and IFNg in combination, however upon every attempts despite using pro-inflammatory induction (LPS and IFNg), the FACS results shows macrophages expressing CD206 (M2 marker) higher compared to CD86 (M1 marker). Upon obtaining the pellet of PBMC layer, the cell pellet are resuspended with SFM and seeded into wells for cell attachment for about 3 hours. Upon cell attachment, the SFM are discarded and M-CSF medium (50ng/ml) is added into the wells (for macrophage differentiation) and left for 5 days without medium change. On the 6th day, the medium is discarded and a medium comprising 50ng/ml of MSCF, 50ng/ml of IFNg and 10ng/ml of LPS is added into the well and left for 48 hours. After that, the cells are detached and stained with CD14, CD206 and CD80 together as mixed stain and later, analyzed with FACS. The MFI value for CD206 shows to be higher in comparison to CD80 MFI value. I have also used varying concentration combination of LPS and IFNg, using the same incubation period as mentioned above, yet encounter the same issue at every attempt (CD206 is highly expressed compared to CD80). In all the attempts, M-CSF concentration was fixed to 50ng/ml. I would like to know which point is misleading.

Thank you for your help in advance.

I want to polarize THP-1 to M0, then to M2. After adding PMA and getting macrophages, I need to get the highest M2 percentage (90%) because I can not isolate M2 from other subtypes. For this reason, Does IL-4 alone enough to polarize macrophages to M2 or I need to use other cytokines like IL-13 or IL-10? in addition, what is the most practical CD marker for M2? CD206 or CD163? there are many researchers who say that it is not easy to get CD206 so should I use CD163 instead?

Thank you

I treat THP1 cells with 100 ng/ml PMA for 48 hrs and then stimulate them with 20 ng/ml of IL-4 and IL-13 for 48hrs (M2) and with 20 ng/ml IFN-gamma and 10 ng/ml LPS for 48hrs (M1). Then I leave them in fresh media for another 2-3 days. I use accuatse to detach them for FACs analysis. while CD11b confirmed monocyte to macrophage, I get negative population for CD206 in M2 macrophages and HLA-DR in M1 macrophages. I would appreciate if anyone can share experience with thp1 polarization and FACs. I do see evident morphological change, but thats not translating to my facs analysis.

Thank you.

Looking forward to hearing suggestions.

I have recently started to culture RAW 264.7 cells. And I used IL-4 to induce RAW264.7 to differentiate into M2 macrophage. Bue the WB result showed the CD206 was very low.