I am differentiating THP1 cells to M2 macrophages using PMA anf IL-4 and IL-13. For THP1 cells I have used RPMI@ATCC media with 10% FBS, 1% anti-anti and 0.05mM beta marcaptoethanol. I have tried different strategies to differntiate but the best I could get 25% expression of M2 macrophages.
I have used 12 well plates, with 500,000 cells/ml/well, differentiate with 150 nM PMA for 24 hours and then 20ng/ml interleukins for 48 hrs. Also changed the duration of interleukins treatment to 48hrs 2 times. Then changed the PMA concentration to 100nM for around 72 hours interleukins for 48hrs+48hrs. Also tried to seed them at 300,000 cells/ml in 24 well plates.
Can anyone help me with a detail protocol for THP1 differentiation to M2 macrophages.
I did flowcytomerty to confirm the presence of M2 macrophages using CD-206 marker .
Hi Samsad,
Check this paper out. The protocol here described is slightly different to the one you have used.
Article The phorbol 12-myristate-13-acetate differentiation protocol...
I hope this helps.
Phorbol 12-myristate 13-acetate (PMA) is a classic protein kinase C (PKC) activator, and its mechanism of inducing THP-1 cells to differentiate into macrophages has been widely confirmed. In cell differentiation studies, PMA activates the PKC signaling pathway to promote the transformation of THP-1 cells from suspended monocytes to adherent macrophages.
The high-purity PMA (≥99% purity, Cat.No.: HY-18739) provided by MCE (MedChemExpress) has been verified by system biology: at a concentration gradient of 100 ng/mL and 200 ng/mL, treatment for 24 hours and 48 hours can effectively induce THP-1 cells to differentiate into macrophage-like cells.
Related studies also detected the expression of macrophage surface markers (such as CD11b, CD68) by flow cytometry, and combined with the analysis of the secretion level of inflammatory factors (such as TNF-α, IL-6), confirming the effectiveness of the differentiation model.
The specific experimental data can be found in the following DOI (data provided by the MCE experimental team):
DOI: 10.13140/RG.2.2.23477.79845
Experiment Findings [Finding] Phorbol 12-myristate 13-acetate (PMA) Induces Diff...
For specific experimental methods and experimental precautions, please refer to the following web link, or contact the MCE technical team for professional after-sales consultation.
Phorbol 12-myristate 13-acetate (PMA)-induction THP-1 protocol:
https://www.medchemexpress.com/Phorbol-12-myristate-13-acetate.html
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