Comparative analysis of RT-qPCR, flow cytometry, and Di-4-ANEPPDHQ fluorescence for distinguishing macrophages phenotypes

This study evaluates the effectiveness of fluorescence microscopy using Di-4-ANEPPDHQ in differentiating macrophage phenotypes (M0, M1, and M2) compared to RT-qPCR and flow cytometry. Using THP-1 monocyte-derived macrophages, we assessed cytokine expression (IL-1β, IL-6, IL-10) via RT-qPCR, surface markers (CD86, CD64, CD206) through flow cytometry, and membrane properties with Di-4-ANEPPDHQ fluorescence. RT-qPCR showed significant differences in cytokine expression: M1 macrophages had elevated IL-1β (p < 0.0001) and IL-6 (p < 0.0001), while M2 macrophages exhibited higher IL-10 levels (p = 0.0030). Flow cytometry revealed distinct surface marker profiles, with M1 expressing high CD64 and M2 showing increased CD206. Di-4-ANEPPDHQ fluorescence indicated membrane order differences: M1 macrophages were depolarized (red shift), while M2 macrophages were hyperpolarized (blue shift). Statistical analysis confirmed high sensitivity and specificity for RT-qPCR and flow cytometry, while Di-4-ANEPPDHQ fluorescence technique provides real-time observations of changes in macrophage membrane behavior, enhancing understanding of their dynamic properties under various conditions. These findings highlight the value of integrating these methods for comprehensive macrophage phenotype characterization, which can aid in understanding macrophage polarization in immune responses and disease contexts.