Hello everyone,
I need to help for the differentiation of my Thp1.
I use 20ng / ml of PMA for 72h to transform Thp1 macrophage adherent.
Then I polarize them in M1 with LPS 15ng / ml and IFNg 50ng / ml, or in M2 with IL13 and Il4 at 25ng / ml, during 48h.
Then I want to characterize them in flow cytometry.
I use the markers:
M1: CD80, CD68 and iNos (intracellular)
M2: CD163, CD206 and Arg1 (intracellular)
But I do not get any results. All my markings are negative ... (only Inos seems to be positive in M1 and M2 permeabilized ...)
PS : To take off the support, I used several techniques: scraping, EDTA 5mM or trypsin 0.05%. Only tryspine managed to take off the cells without destroying them ...
Do you have any tips or ideas on my problem?
Thank you
THP-1 cells are macrophage-like when they are differentiated by PMA, but are not completely like macrophages and may not express classic M1 and M2 markers at the same levels as on primary macrophages.
I would like to point out that using trypsin to detach your cells will cleave any of the external markers you are trying to look at. Based on your panel, I am not entirely sure that I would expect to see much of anything except CD68. Also worth noting, to detect CD68 you must permeabilize the cells.
Here is a useful manual from BD Biosciences attached to this answer. If you go to page 11, they have a list of markers and what kind of expression you should expect from THP-1 cells. Most of the markers you have listed do not appear in this chart to be expressed (although they are not using IFNg/LPS or IL13/IL4.
In case the site takes down the manual here is the original link to the pdf.
https://www.bdbiosciences.com/documents/BD_Multicolor_MonocyteMacrophageDiff_AppNote.pdf
Hi,
Thp-1 cell line is good only to start working with monocyte-like cells and learn the basic techniques. As Jared mentioned they do not express many markers which are typical for monocytes/macrophages. Also depending on the cPD (cumulative population doubling) they decrease expression of many proteins and antigens. They need stronger impulse to induce differentiation than primary monocytes. There are simply too many differences between this cell line and primary monocytes...
If is your research focused primary on monocytes/macrophages I would recommend to use isolated primary monocytes and not wasting time with Thp-1 cell line.
hope this helps a little
Hello,
Thank you for your help.
@Jared Paul Taylor, The problem with the Bioscience manual is that they make the difference between Monocyte and Macrophage. But I would like to make the difference between M1 and M2.
My current markers come from various articles using Thp1. But indeed, there are surely current markers that are not expressed by Thp1.
For trypsin, I agree with you. But all my other methods have been a failure: EDTA 5mM cells do not come off, and scraping I have a lot of debris ...
@Matej Vicen. Yes, we will go through PBMCs later.
But we want to use Thp1 to test plasmid mutation effects.
My next step is to try to characterize this M1 / M2 differentiation via ELISA or RT-qPCR.
Tourneur-Marsille Julien
One method you can try is using UpCell plates:
https://www.thermofisher.com/order/catalog/product/174902
These are plates that I have used in the past for primary monocyte-derived macrophages. The macrophages are adherent at 37 degrees. However, when you cool the plates in the 4 degree the material changes its hydrophilicity allowing the cells to detach. This method works very well for primary monocyte-derived macrophages which stick to the plates much stronger than THP-1. Alternatively, you can use ultra-low cluster tissue culture plates which are cheaper than UpCell. You can usually get lightly adherent cells off with gentle rinsing and avoid using enzymatic reagents.
ELISA method you suggested might be better for this particular type of characterization.
Hi Tourneur-Marsille Julien ,
Were you able to resolve the issue? I am facing a similar issue with THP1 macrophage polarization. I treat monocytes with 100 ng/ml PMA for 48hrs, and stimulate them with 20ng/ml IL4 and IL-13 for 48hrs towards M2 and 10 ng/ml LPS and 20ng/ml IFN-gamma for 48hrs towards M1. Then I would leave them in fresh media without stimulants for 2-3 days. I used CD11b to confirm differentiation, which was positive for me. But CD206 for M2 and HLA-DR for M1 was negative.
Thanks
Hello,
No, I did not manage to detect CD206 in my differentiated Thp1.
But I found this article that specifies that Thp1 do not express CD206 when differentiated with PMA. But they express it if they are by MDM.
I think the best way to evaluate their differentiation is to study their secreted cytokine.
Personally, we dropped the Thp1, and start on PBMCs.
Good luck
"CD206 was detected in MDM in approximately half of the MDM population but not the PMAr THP-1 cells. Macrophages can sense the microenvironment they are exposed to and adapt polarity "
https://www-ncbi-nlm-nih-gov.gate2.inist.fr/pmc/articles/PMC2800192/
doi: 10.1371/journal.pone.0008668
Hello,
Yes. PMA treated THP1 wont show CD206 because they are just M0 and not M2. I dont see any positive for CD206 even when they are polairsed to M2.
Thank you !
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