ArticleBiochemistry and biophysics reports2025
Comparative analysis of RT-qPCR, flow cytometry, and Di-4-ANEPPDHQ fluorescence for distinguishing macrophages phenotypes.
Article in Biochemistry and biophysics reports, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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Who cites it
2 citing papers in PubMed.
- Disorder in Order-Related Membrane Biophysical Parameters: An In-Depth Analysis of Di-4-ANEPPDHQ Generalized Polarization.The journal of physical chemistry letters · 2026Article
- The Interaction BetweenPathogens (Basel, Switzerland) · 2026Article
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Authors and funding
7 authors.
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Abstract
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.
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