An Optimized Protocol for the Differentiation and Fibrosis-Associated Transcriptional Priming of THP-1 Macrophages

Monocyte-to-macrophage differentiation and subsequent activation play a crucial role in tissue fibrosis. While the human monocytic cell line THP-1 is widely used as an in vitro model, variations in the use of phorbol 12-myristate 13-acetate (PMA) differentiation often led to inconsistent macrophage phenotypes. Here, we present an optimized protocol yielding mature differentiated THP-1 macrophages (d-THP-1) with stable baseline competence for fibrosis-associated transcriptional change. A protocol consisting of 48 h PMA treatment followed by a 24 h resting phase successfully generated macrophage-like THP-1 cells, confirmed by microscopic assessment and flow cytometric analysis of surface markers. Furthermore, the responsiveness of these optimized macrophages to the chemokine CXCL12 was validated. Following stimulation, the optimized cells exhibited a potentially fibrosis-associated state, characterized by significant early upregulation of matrix metalloproteinase 9 (MMP9) and late upregulation of transforming growth factor-β (TGF-β) measured by RT-qPCR. This reproducible protocol provides a reliable in vitro platform for studying macrophage-mediated fibrotic pathways and screening of therapeutic targets.