Altuntaş C., Duruksu G., Hunç F., Burhanoğlu T., Furat S. H.
Acta Medica Nicomedia, vol.9, pp.1-11, 2026 (TRDizin)
Abstract
Objective: This study aims to investigate the differentiation of the U937 cell line into M1-polarized macrophages using four protocols described in previous studies, but which have not been standardized on account of phenotypic plasticity and functional instability of macrophages. The resulting phenotypic alternations were characterized. By revealing the flow cytometric profiles of the M1-polarized macrophages, obtained by implementation of different chemical induction protocols on the cells, this study provides invaluable data on the use of in vitro models involving M1 polarized macrophages. Methods: Four different chemical induction strategies were implemented on U937 cells to obtain M1-polarized macrophages, each involving either phorbol-12-myristate-13-acetate (PMA) alone or combine with additional stimulants such as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). After differentiation, the expression of the macrophage-specific surface markers CD68 (M1) and CD206 (M2) was analyzed by flow cytometry, and the polarization trends were examined based on the expression of the associated markers. Results: A divergence in the M1-macrophage-specific cell surface marker was observed among the applied macrophage differentiation strategies. This finding indicates that the balance between pro-inflammatory (M1-like) and anti-inflammatory (M2-like) phenotypes is subject to variation depending on the applied protocol. Furthermore, differentiation strategies (2) yielded optimal results in terms of cells exhibiting the M1 polarized macrophage phenotype (61%). Conclusion: The findings indicate that non-standardized differentiation protocols result in the generation of distinct macrophage-like populations from U937 cells regarding polarization. These findings emphasize the importance of standardization in experimental designs and provide a framework for selecting appropriate differentiation methods in studies of macrophage biology.