Hi ALL,
I constantly differentiate human macrophage from THP-1 cell line using 100 ng/mL PMA (phorbol 12-myristate 13-acetate) for only 24h (DAY 1), then change the media (RPMI) and leave them rest for 24h (DAY 2). At DAY 3, they are ready to seed into a well plate containing testing agents. However, as my observation, the most abundant population is M0, which is non-active phenotype, together with few of M1 and M2 phenotypes. I desire to fully differentiate them into M1 and M2, should I differentiate them for 48h at DAY 1 instead of 24h? Any comments would be much appreciated!
In our lab, we have this protocol for THP-1 differentiation. Add PMA at 50 ng/mL, incubate cells for three days. On the fourth day, wash cells and allow to rest in PMA-free media for a minimum of 24 h before analysis.
Thien Ngoc Le , hello!
About the 24-hour time-effect analysis of 100 ng/mL PMA in vitro differentiation of human dTHP-1 macrophages:
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 phenotype transformation of THP-1 cells from suspended monocytes to adherent macrophages.
According to the experimental conditions you mentioned, 100 ng/mL PMA treatment for 24 hours is sufficient to induce dTHP-1 cells to complete macrophage differentiation.
- The high-purity PMA (≥99% purity, Cat.No.: HY-18739) provided by MCE (MedChemExpress) has been verified by system biology and shows that 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 tested 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.
- Specific experimental data can be viewed through the following DOI (data provided by the MCE experimental team).
References:
Experiment Findings [Finding] Phorbol 12-myristate 13-acetate (PMA) Induces Diff...
- DOI: 10.13140/RG.2.2.23477.79845
- https://www.medchemexpress.com/Phorbol-12-myristate-13-acetate.html