I would like to be able to differentiate THP-1 into macrophages of several types, in particular M1 and M2a.
To do this, I activated THP-1 cells with 200 ng/mL PMA for 72h, than I differentiated them with 48h of cytokines (GM-CSF and IFNg for M1, M-CSF and IL4 for M2a). I was satisfied when I saw the appearance of the cells, which looked like what I observe when using monocytes and not THP-1.
Nevertheless, once passed through the flow cytometer, my cells do not express any of the markers I was expecting (CD68, CD163, CD80 ...).
I don't know what to do because I already tried different protocols with different cell/PMA concentration and activation time ... And this protocol seemed to me to be the most suitable ...
What can I do ??
Thanks !
Zélie Dirand,
PhD student
Hello,
What is the passage # of your cells. If THP-1 has undergone many passages or if they are just left to become over confluent, there is a high likelihood that the cells could change their phenotype. Usually, you should use THP-1 cells below passage 30.
Another possibility could be the culture conditions such as concentration and activation time before starting differentiation studies in THP-1 cells. The heterogeneity of the culture could affect the expression levels of markers.
Please refer to the research article given below.
https://doi.org/10.1111/aji.12129
Best.
The passage is relatively low, I am at P10. I have been careful not to let my cells become too confluent, and I have recovered 2 THP-1 lines from 2 different colleagues (and my results are the same with both lines).
Thank you for the article !
First, the concentration of PMA being used is at least 4 times higher than needed. 50ng or 125nM is the recommended conc. which also worked well in my pair of hands.
Second, THP-1 cells stimulated with PMA at that recommended conc. will be differentiated within 24-48h. So, 72h stimulation with 200ng PMA is harsh !
Coming back to expression markers detection by flow, PMA stimulated THP-1 cells adhere more strongly to the surface and using trypsin or ETDA to detach cells will cleave any of the external markers you are trying to look at. So, its not a surprise that you failed to see any expression of those markers. Alternatively, try imaging the cells after staining with Abs of your choice. You can also try western blotting or RT-PCR
BW
I seeded THP-1 on glass slides last week to perform immuno-fluorescence, so I will try this afternoon with the same markers as in RT-qPCR. I hope it will be good, than you much for your answer ! I wouldn't have thought that trypsin could remove the external markers ... When thinking about it, it seems quite logical.
Zélie
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